Endonuclease-substrate complexes

ABSTRACT

The present invention provides novel cleavage agents for the cleavage and modification of nucleic acid. The cleavage agents find use, for example, for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. In some embodiments, an endonuclease activity is used to cleave a target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

The present application claims priority to U.S. Provisional ApplicationSer. No. 60/448,601, filed Feb. 20, 2003, and U.S. ProvisionalApplication Ser. No. 60/452,008, filed Mar. 4, 2003, each of which isincorporated herein by reference. The present application alsoincorporates by reference co-pending U.S. patent application Ser. No.09/714,935, filed Nov. 17, 2000, U.S. patent application Ser. No.10/290,386, filed Nov. 11, 2002, and PCT Application No. PCT/US01/44953,filed Nov. 15, 2001, each in their entirety.

FIELD OF THE INVENTION

The present invention provides novel cleavage agents and polymerases forthe cleavage and modification of nucleic acid. The cleavage agents andpolymerases find use, for example, for the detection andcharacterization of nucleic acid sequences and variations in nucleicacid sequences. In some embodiments, the 5′ nuclease activity of avariety of enzymes is used to cleave a target-dependent cleavagestructure, thereby indicating the presence of specific nucleic acidsequences or specific variations thereof.

BACKGROUND OF THE INVENTION

Methods for the detection and characterization of specific nucleic acidsequences and sequence variations have been used to detect the presenceof viral or bacterial nucleic acid sequences indicative of an infection,to detect the presence of variants or alleles of genes associated withdisease and cancers. These methods also find application in theidentification of sources of nucleic acids, as for forensic analysis orfor paternity determinations.

Various methods are known to the art that may be used to detect andcharacterize specific nucleic acid sequences and sequence variants.Nonetheless, with the completion of the nucleic acid sequencing of thehuman genome, as well as the genomes of numerous pathogenic organisms,the demand for fast, reliable, cost-effective and user-friendly testsfor the detection of specific nucleic acid sequences continues to grow.Importantly, these tests must be able to create a detectable signal fromsamples that contain very few copies of the sequence of interest. Thefollowing discussion examines two levels of nucleic acid detectionassays currently in use: I. Signal Amplification Technology fordetection of rare sequences; and II. Direct Detection Technology forquantitative detection of sequences.

I. Signal Amplification Technology Methods for Amplification

The “Polymerase Chain Reaction” (PCR) comprises the first generation ofmethods for nucleic acid amplification. However, several other methodshave been developed that employ the same basis of specificity, butcreate signal by different amplification mechanisms. These methodsinclude the “Ligase Chain Reaction” (LCR), “Self-Sustained SyntheticReaction” (3SR/NASBA), and “Qβ-Replicase” (Qβ).

Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR), as described in U.S. Pat. Nos.4,683,195, 4,683,202, and 4,965,188 to Mullis and Mullis et al. (thedisclosures of which are hereby incorporated by reference), describe amethod for increasing the concentration of a segment of target sequencein a mixture of genomic DNA without cloning or purification. Thistechnology provides one approach to the problems of low target sequenceconcentration. PCR can be used to directly increase the concentration ofthe target to an easily detectable level. This process for amplifyingthe target sequence involves introducing a molar excess of twooligonucleotide primers that are complementary to their respectivestrands of the double-stranded target sequence to the DNA mixturecontaining the desired target sequence. The mixture is denatured andthen allowed to hybridize. Following hybridization, the primers areextended with polymerase so as to form complementary strands. The stepsof denaturation, hybridization, and polymerase extension can be repeatedas often as needed, in order to obtain relatively high concentrations ofa segment of the desired target sequence.

The length of the segment of the desired target sequence is determinedby the relative positions of the primers with respect to each other,and, therefore, this length is a controllable parameter. Because thedesired segments of the target sequence become the dominant sequences(in terms of concentration) in the mixture, they are said to be“PCR-amplified.”

Ligase Chain Reaction (LCR or LAR)

The ligase chain reaction (LCR; sometimes referred to as “LigaseAmplification Reaction” (LAR) described by Barany, Proc. Natl. Acad.Sci., 88:189 (1991); Barany, PCR Methods and Applic., 1:5 (1991); and Wuand Wallace, Genomics 4:560 (1989) has developed into a well-recognizedalternative method for amplifying nucleic acids. In LCR, fouroligonucleotides, two adjacent oligonucleotides which uniquely hybridizeto one strand of target DNA, and a complementary set of adjacentoligonucleotides, that hybridize to the opposite strand are mixed andDNA ligase is added to the mixture. Provided that there is completecomplementarity at the junction, ligase will covalently link each set ofhybridized molecules. Importantly, in LCR, two probes are ligatedtogether only when they base-pair with sequences in the target sample,without gaps or mismatches. Repeated cycles of denaturation,hybridization and ligation amplify a short segment of DNA. LCR has alsobeen used in combination with PCR to achieve enhanced detection ofsingle-base changes. Segev, PCT Public. No. W09001069 A1 (1990).However, because the four oligonucleotides used in this assay can pairto form two short ligatable fragments, there is the potential for thegeneration of target-independent background signal. The use of LCR formutant screening is limited to the examination of specific nucleic acidpositions.

Self-Sustained Synthetic Reaction (3SR/NASBA)

The self-sustained sequence replication reaction (3SR) (Guatelli et al.,Proc. Natl. Acad. Sci., 87:1874-1878 [1990], with an erratum at Proc.Natl. Acad. Sci., 87:7797 [1990]) is a transcription-based in vitroamplification system (Kwok et al., Proc. Natl. Acad. Sci., 86:1173-1177[1989]) that can exponentially amplify RNA sequences at a uniformtemperature. The amplified RNA can then be utilized for mutationdetection (Fahy et al., PCR Meth. Appl., 1:25-33 [1991]). In thismethod, an oligonucleotide primer is used to add a phage RNA polymerasepromoter to the 5′ end of the sequence of interest. In a cocktail ofenzymes and substrates that includes a second primer, reversetranscriptase, RNase H, RNA polymerase and ribo-and deoxyribonucleosidetriphosphates, the target sequence undergoes repeated rounds oftranscription, cDNA synthesis and second-strand synthesis to amplify thearea of interest. The use of 3SR to detect mutations is kineticallylimited to screening small segments of DNA (e.g., 200-300 base pairs).

Q-Beta (Qβ) Replicase

In this method, a probe that recognizes the sequence of interest isattached to the replicatable RNA template for Qβ replicase. A previouslyidentified major problem with false positives resulting from thereplication of unhybridized probes has been addressed through use of asequence-specific ligation step. However, available thermostable DNAligases are not effective on this RNA substrate, so the ligation must beperformed by T4 DNA ligase at low temperatures (37° C.). This preventsthe use of high temperature as a means of achieving specificity as inthe LCR, the ligation event can be used to detect a mutation at thejunction site, but not elsewhere.

Table 1 below, lists some of the features desirable for systems usefulin sensitive nucleic acid diagnostics, and summarizes the abilities ofeach of the major amplification methods (See also, Landgren, Trends inGenetics 9:199 [1993]).

A successful diagnostic method must be very specific. A straight-forwardmethod of controlling the specificity of nucleic acid hybridization isby controlling the temperature of the reaction. While the 3SR/NASBA, andQβ systems are all able to generate a large quantity of signal, one ormore of the enzymes involved in each cannot be used at high temperature(i.e., >55° C.). Therefore the reaction temperatures cannot be raised toprevent non-specific hybridization of the probes. If probes areshortened in order to make them melt more easily at low temperatures,the likelihood of having more than one perfect match in a complex genomeincreases. For these reasons, PCR and LCR currently dominate theresearch field in detection technologies.

TABLE 1 Method PCR & 3SR Feature PCR LCR LCR NASBA Qβ AmplifiesTarget + + + + Recognition of Independent + + + + + Sequences RequiredPerformed at High Temp. + + Operates at Fixed Temp. + + ExponentialAmplification + + + + + Generic Signal Generation + Easily Automatable

The basis of the amplification procedure in the PCR and LCR is the factthat the products of one cycle become usable templates in all subsequentcycles, consequently doubling the population with each cycle. The finalyield of any such doubling system can be expressed as: (1+X)^(n)=y,where “X” is the mean efficiency (percent copied in each cycle), “n” isthe number of cycles, and “y” is the overall efficiency, or yield of thereaction (Mullis, PCR Methods Applic., 1:1 [1991]). If every copy of atarget DNA is utilized as a template in every cycle of a polymerasechain reaction, then the mean efficiency is 100%. If 20 cycles of PCRare performed, then the yield will be 2²⁰, or 1,048,576 copies of thestarting material. If the reaction conditions reduce the mean efficiencyto 85%, then the yield in those 20 cycles will be only 1.85²⁰, or220,513 copies of the starting material. In other words, a PCR runningat 85% efficiency will yield only 21% as much final product, compared toa reaction running at 100% efficiency. A reaction that is reduced to 50%mean efficiency will yield less than 1% of the possible product.

In practice, routine polymerase chain reactions rarely achieve thetheoretical maximum yield, and PCRs are usually run for more than 20cycles to compensate for the lower yield. At 50% mean efficiency, itwould take 34 cycles to achieve the million-fold amplificationtheoretically possible in 20, and at lower efficiencies, the number ofcycles required becomes prohibitive. In addition, any backgroundproducts that amplify with a better mean efficiency than the intendedtarget will become the dominant products.

Also, many variables can influence the mean efficiency of PCR, includingtarget DNA length and secondary structure, primer length and design,primer and dNTP concentrations, and buffer composition, to name but afew. Contamination of the reaction with exogenous DNA (e.g., DNA spilledonto lab surfaces) or cross-contamination is also a major consideration.Reaction conditions must be carefully optimized for each differentprimer pair and target sequence, and the process can take days, even foran experienced investigator. The laboriousness of this process,including numerous technical considerations and other factors, presentsa significant drawback to using PCR in the clinical setting. Indeed, PCRhas yet to penetrate the clinical market in a significant way. The sameconcerns arise with LCR, as LCR must also be optimized to use differentoligonucleotide sequences for each target sequence. In addition, bothmethods require expensive equipment, capable of precise temperaturecycling.

Many applications of nucleic acid detection technologies, such as instudies of allelic variation, involve not only detection of a specificsequence in a complex background, but also the discrimination betweensequences with few, or single, nucleotide differences. One method forthe detection of allele-specific variants by PCR is based upon the factthat it is difficult for Taq polymerase to synthesize a DNA strand whenthere is a mismatch between the template strand and the 3′ end of theprimer. An allele-specific variant may be detected by the use of aprimer that is perfectly matched with only one of the possible alleles;the mismatch to the other allele acts to prevent the extension of theprimer, thereby preventing the amplification of that sequence. Thismethod has a substantial limitation in that the base composition of themismatch influences the ability to prevent extension across themismatch, and certain mismatches do not prevent extension or have only aminimal effect (Kwok et al., Nucl. Acids Res., 18:999 [1990]).)

A similar 3′-mismatch strategy is used with greater effect to preventligation in the LCR (Barany, PCR Meth. Applic., 1:5 [1991]). Anymismatch effectively blocks the action of the thermostable ligase, butLCR still has the drawback of target-independent background ligationproducts initiating the amplification. Moreover, the combination of PCRwith subsequent LCR to identify the nucleotides at individual positionsis also a clearly cumbersome proposition for the clinical laboratory.

II. Direct Detection Technology

When a sufficient amount of a nucleic acid to be detected is available,there are advantages to detecting that sequence directly, instead ofmaking more copies of that target, (e.g., as in PCR and LCR). Mostnotably, a method that does not amplify the signal exponentially is moreamenable to quantitative analysis. Even if the signal is enhanced byattaching multiple dyes to a single oligonucleotide, the correlationbetween the final signal intensity and amount of target is direct. Sucha system has an additional advantage that the products of the reactionwill not themselves promote further reaction, so contamination of labsurfaces by the products is not as much of a concern. Traditionalmethods of direct detection including Northern and Southern blotting andRNase protection assays usually require the use of radioactivity and arenot amenable to automation. Recently devised techniques have sought toeliminate the use of radioactivity and/or improve the sensitivity inautomatable formats. Two examples are the “Cycling Probe Reaction”(CPR), and “Branched DNA” (bDNA)

The cycling probe reaction (CPR) (Duck et al., BioTech., 9:142 [1990]),uses a long chimeric oligonucleotide in which a central portion is madeof RNA while the two termini are made of DNA. Hybridization of the probeto a target DNA and exposure to a thermostable RNase H causes the RNAportion to be digested. This destabilizes the remaining DNA portions ofthe duplex, releasing the remainder of the probe from the target DNA andallowing another probe molecule to repeat the process. The signal, inthe form of cleaved probe molecules, accumulates at a linear rate. Whilethe repeating process increases the signal, the RNA portion of theoligonucleotide is vulnerable to RNases that may be carried throughsample preparation.

Branched DNA (bDNA), described by Urdea et al., Gene 61:253-264 (1987),involves oligonucleotides with branched structures that allow eachindividual oligonucleotide to carry 35 to 40 labels (e.g., alkalinephosphatase enzymes). While this enhances the signal from ahybridization event, signal from non-specific binding is similarlyincreased.

While both of these methods have the advantages of direct detectiondiscussed above, neither the CPR or bDNA methods can make use of thespecificity allowed by the requirement of independent recognition by twoor more probe (oligonucleotide) sequences, as is common in the signalamplification methods described in Section I. above. The requirementthat two oligonucleotides must hybridize to a target nucleic acid inorder for a detectable signal to be generated confers an extra measureof stringency on any detection assay. Requiring two oligonucleotides tobind to a target nucleic acid reduces the chance that false “positive”results will be produced due to the non-specific binding of a probe tothe target. The further requirement that the two oligonucleotides mustbind in a specific orientation relative to the target, as is required inPCR, where oligonucleotides must be oppositely but appropriatelyoriented such that the DNA polymerase can bridge the gap between the twooligonucleotides in both directions, further enhances specificity of thedetection reaction. However, it is well known to those in the art thateven though PCR utilizes two oligonucleotide probes (termed primers)“non-specific” amplification (i.e., amplification of sequences notdirected by the two primers used) is a common artifact. This is in partbecause the DNA polymerase used in PCR can accommodate very largedistances, measured in nucleotides, between the oligonucleotides andthus there is a large window in which non-specific binding of anoligonucleotide can lead to exponential amplification of inappropriateproduct. The LCR, in contrast, cannot proceed unless theoligonucleotides used are bound to the target adjacent to each other andso the full benefit of the dual oligonucleotide hybridization isrealized.

An ideal direct detection method would combine the advantages of thedirect detection assays (e.g., easy quantification and minimal risk ofcarry-over contamination) with the specificity provided by a dualoligonucleotide hybridization assay.

SUMMARY OF THE INVENTION

The present invention provides novel cleavage agents for the cleavageand modification of nucleic acids. The cleavage agents find use, forexample, for the detection and characterization of nucleic acidsequences and variations in nucleic acid sequences. In some embodiments,the 5′ nuclease activity of a variety of enzymes is used to cleave atarget-dependent cleavage structure, thereby indicating the presence ofspecific nucleic acid sequences or specific variations thereof. Thepresent invention contemplates use of novel detection methods forvarious uses, including, but not limited to, clinical diagnosticpurposes.

The present invention provides structure-specific cleavage agents (e.g.,nucleases) from a variety of sources, including mesophilic,psychrophilic, thermophilic, and hyperthermophilic organisms. Thepreferred structure-specific nucleases are thermostable. Thermostablestructure-specific nucleases are contemplated as particularly useful inthat they operate at temperatures where nucleic acid hybridization isextremely specific, allowing for allele-specific detection (includingsingle-base mismatches). In one embodiment, the thermostablestructure-specific nucleases are thermostable 5′ nucleases comprisingaltered polymerases derived from the native polymerases of Thermusspecies, including, but not limited to Thermus aquaticus, Thermusflavus, and Thermus thermophilus. However, the invention is not limitedto the use of thermostable 5′ nucleases. Thermostable structure-specificnucleases from the FEN-1, RAD2 and XPG class of nucleases are alsopreferred.

The present invention provides a method for detecting a target sequence(e.g., a mutation, polymorphism, etc), comprising providing a samplesuspected of containing the target sequence; oligonucleotides capable offorming an invasive cleavage structure in the presence of the targetsequence; and an agent for detecting the presence of an invasivecleavage structure; and exposing the sample to the oligonucleotides andthe agent. In some embodiments, the method further comprises the step ofdetecting a complex comprising the agent and the invasive cleavagestructure (directly or indirectly). In some embodiments, the agentcomprises a cleavage agent. In some preferred embodiments, the exposingof the sample to the oligonucleotides and the agent comprises exposingthe sample to the oligonucleotides and the agent under conditionswherein an invasive cleavage structure is formed between the targetsequence and the oligonucleotides if the target sequence is present inthe sample, wherein the invasive cleavage structure is cleaved by thecleavage agent to form a cleavage product. In some embodiments, themethod further comprises the step of detecting the cleavage product. Insome embodiments, the target sequence comprises a first region and asecond region, the second region downstream of and contiguous to thefirst region, and wherein the oligonucleotides comprise first and secondoligonucleotides, wherein at least a portion of the firstoligonucleotide is completely complementary to the first portion of thetarget sequence and wherein the second oligonucleotide comprises a 3′portion and a 5′ portion, wherein the 5′ portion is completelycomplementary to the second portion of said target nucleic acid.

The present invention also provides a kit for detecting such targetsequences, said kit comprising oligonucleotides capable of forming aninvasive cleavage structure in the presence of the target sequence. Insome embodiments, the kit further comprises an agent for detecting thepresence of an invasive cleavage structure (e.g., a cleavage agent). Insome embodiments, the oligonucleotides comprise first and secondoligonucleotides, said first oligonucleotide comprising a 5′ portioncomplementary to a first region of the target nucleic acid and saidsecond oligonucleotide comprising a 3′ portion and a 5′ portion, said 5′portion complementary to a second region of the target nucleic aciddownstream of and contiguous to the first portion. In some preferredembodiments, the target sequence comprises human cytomegalovirus viralDNA; sequence containing polymorphisms in the human apolipoprotein Egene (ApoE); sequence containing mutations in the human hemochromatosis(HH) gene; sequence containing mutations in human MTHFR; sequencecontaining prothrombin 20210GA polymorphism; sequence containing HR-2mutation in human factor V gene; sequence containing single nucleotidepolymorphisms in human TNF-α gene, and sequence containing the Leidenmutation in human factor V gene. In some preferred embodiments, kitscomprise oligonucleotides for detecting two or more target sequences.For example, information on two or more mutations may provide medicallyrelevant information such that kits allowing detection of the pluralityof mutations would be desired (e.g., Factor V and HR-2 detection). Insome preferred embodiments kits are probed containing a probeoligonucleotide comprising a sequence of SEQ ID NOs: 197, 198, 199, 200,208, 209, 211, 212, 217, 218, 223, 224, 229, 232, 236, 237, 241, 242, or244. In still other embodiments, kits provide oligonucleotide sets, thesets including one or more of the oligonucleotides: SEQ ID NOs:195, 197,and 198 for ApoE detection; 196, 199, and 200 for ApoE detection; 202,208, and 209 for HH detection; 203, 211, and 212 for HH detection; 216,217, and 218 for MTHFR detection; 222, 223, and 224 for prothrombinpolymorphism detection; 228, 229, 231, and 232 for HR-2 detection; 235,236, and 237 for TNF-α detection; 240, 241, and 242 for Factor Vdetection; and 243, 244, 246, and 247 for MRSA detection.

The present invention further provides detection assay panels comprisingan array of different detection assays. The detection assays includeassays for detecting mutations in nucleic acid molecules and fordetecting gene expression levels. Assays find use, for example, in theidentification of the genetic basis of phenotypes, including medicallyrelevant phenotypes and in the development of diagnostic products,including clinical diagnostic products.

The present invention also provides methods for detecting the presenceof a target nucleic acid molecule by detecting non-target cleavageproducts comprising providing: a cleavage agent; a source of targetnucleic acid, the target nucleic acid comprising a first region and asecond region, the second region downstream of and contiguous to thefirst region; a first oligonucleotide, wherein at least a portion of thefirst oligonucleotide is completely complementary to the first portionof the target nucleic acid; and a second oligonucleotide comprising a 3′portion and a 5′ portion, wherein the 5′ portion is completelycomplementary to the second portion of the target nucleic acid; mixingthe cleavage agent, the target nucleic acid, the first oligonucleotideand the second oligonucleotide to create a reaction mixture underreaction conditions such that at least the portion of the firstoligonucleotide is annealed to the first region of said target nucleicacid and wherein at least the 5′ portion of the second oligonucleotideis annealed to the second region of the target nucleic acid so as tocreate a cleavage structure, and wherein cleavage of the cleavagestructure occurs to generate non-target cleavage product; and detectingthe cleavage of the cleavage structure.

The detection of the cleavage of the cleavage structure can be carriedout in any manner. In some embodiments, the detection of the cleavage ofthe cleavage structure comprises detecting the non-target cleavageproduct. In yet other embodiments, the detection of the cleavage of thecleavage structure comprises detection of fluorescence, mass, orfluorescence energy transfer. Other detection methods include, but arenot limited to detection of radioactivity, luminescence,phosphorescence, fluorescence polarization, and charge. In someembodiments, detection is carried out by a method comprising providingthe non-target cleavage product; a composition comprising twosingle-stranded nucleic acids annealed so as to define a single-strandedportion of a protein binding region; and a protein; and exposing thenon-target cleavage product to the single-stranded portion of theprotein binding region under conditions such that the protein binds tothe protein binding region. In some embodiments, the protein comprises anucleic acid producing protein, wherein the nucleic acid producingprotein binds to the protein binding region and produces nucleic acid.In some embodiments, the protein binding region is a template-dependentRNA polymerase binding region (e.g., a T7 RNA polymerase bindingregion). In other embodiments, the detection is carried out by a methodcomprising providing the non-target cleavage product; a singlecontinuous strand of nucleic acid comprising a sequence defining asingle strand of an RNA polymerase binding region; a template-dependentDNA polymerase; and a template-dependent RNA polymerase; exposing thenon-target cleavage product to the RNA polymerase binding region underconditions such that the non-target cleavage product binds to a portionof the single strand of the RNA polymerase binding region to produce abound non-target cleavage product; exposing the bound non-targetcleavage product to the template-dependent DNA polymerase underconditions such that a double-stranded RNA polymerase binding region isproduced; and exposing the double-stranded RNA polymerase binding regionto the template-dependent RNA polymerase under conditions such that RNAtranscripts are produced. In some embodiments, the method furthercomprises the step of detecting the RNA transcripts. In someembodiments, the template-dependent RNA polymerase is T7 RNA polymerase.

The present invention is not limited by the nature of the 3′ portion ofthe second oligonucleotide. In some preferred embodiments, the 3′portion of the second oligonucleotide comprises a 3′ terminal nucleotidenot complementary to the target nucleic acid. In some embodiments, the3′ portion of the second oligonucleotide consists of a single nucleotidenot complementary to the target nucleic acid.

Any of the components of the method may be attached to a solid support.For example, in some embodiments, the first oligonucleotide is attachedto a solid support. In other embodiments, the second oligonucleotide isattached to a solid support.

The cleavage agent can be any agent that is capable of cleaving invasivecleavage structures. In some preferred embodiments, the cleavage agentcomprises a structure-specific nuclease. In particularly preferredembodiments, the structure-specific nuclease comprises a thermostablestructure-specific nuclease (e.g., a thermostable 5′ nuclease).Thermostable structure-specific nucleases include, but are not limitedto, those having an amino acid sequence homologous to a portion of theamino acid sequence of a thermostable DNA polymerase derived from athermophilic organism (e.g., Thermus aquaticus, Thermus flavus, andThermus thermophilus). In other embodiments, the thermostablestructure-specific nucleases is from the FEN-1, RAD2 or XPG class ofnucleases, a chimerical structures containing one or more portions ofany of the above cleavage agents. In preferred embodiments, the cleavageagent comprises a Y33 equivalent residue.

The method is not limited by the nature of the target nucleic acid. Insome embodiments, the target nucleic acid is single stranded or doublestranded DNA or RNA. In some embodiments, double stranded nucleic acidis rendered single stranded (e.g., by heat) prior to formation of thecleavage structure. In some embodiment, the source of target nucleicacid comprises a sample containing genomic DNA. Sample include, but arenot limited to, blood, saliva, cerebral spinal fluid, pleural fluid,milk, lymph, sputum and semen.

In some embodiments, the reaction conditions for the method compriseproviding a source of divalent cations. In some preferred embodiments,the divalent cation is selected from the group comprising Mn²⁺ and Mg²⁺ions. In some embodiments, the reaction conditions for the methodcomprise providing the first and the second oligonucleotides inconcentration excess compared to the target nucleic acid.

In some embodiments, the method further comprises providing a thirdoligonucleotide complementary to a third portion of said target nucleicacid upstream of the first portion of the target nucleic acid, whereinthe third oligonucleotide is mixed with the reaction mixture.

The present invention also provides a method for detecting the presenceof a target nucleic acid molecule by detecting non-target cleavageproducts comprising providing: a cleavage agent; a source of targetnucleic acid, the target nucleic acid comprising a first region and asecond region, the second region downstream of and contiguous to thefirst region; a plurality of first oligonucleotides, wherein at least aportion of the first oligonucleotides is completely complementary to thefirst portion of the target nucleic acid; a second oligonucleotidecomprising a 3′ portion and a 5′ portion, wherein said 5′ portion iscompletely complementary to the second portion of the target nucleicacid; mixing the cleavage agent, the target nucleic acid, the pluralityof first oligonucleotides and second oligonucleotide to create areaction mixture under reaction conditions such that at least theportion of a first oligonucleotide is annealed to the first region ofthe target nucleic acid and wherein at least the 5′ portion of thesecond oligonucleotide is annealed to the second region of the targetnucleic acid so as to create a cleavage structure, and wherein cleavageof the cleavage structure occurs to generate non-target cleavageproduct, wherein the conditions permit multiple cleavage structures toform and be cleaved from the target nucleic acid; and detecting thecleavage of said cleavage structures. In some embodiments, theconditions comprise isothermal conditions that permit the plurality offirst oligonucleotides to dissociate from the target nucleic acid. Whilethe present invention is limited by the number of cleavage structureformed on a particular target nucleic acid, in some preferredembodiments, two or more (3, 4, 5, . . . , 10, . . . , 10000, . . .) ofthe plurality of first oligonucleotides form cleavage structures with aparticular target nucleic acid, wherein the cleavage structures arecleaved to produce the non-target cleavage products.

The present invention also provide methods where a cleavage product fromthe above methods is used in a further invasive cleavage reaction. Forexample, the present invention provides a method comprising providing acleavage agent; a first target nucleic acid, the first target nucleicacid comprising a first region and a second region, the second regiondownstream of and contiguous to the first region; a firstoligonucleotide, wherein at least a portion of the first oligonucleotideis completely complementary to the first portion of the first targetnucleic acid; a second oligonucleotide comprising a 3′ portion and a 5′portion, wherein the 5′ portion is completely complementary to thesecond portion of the first target nucleic acid; a second target nucleicacid, said second target nucleic acid comprising a first region and asecond region, the second region downstream of and contiguous to thefirst region; and a third oligonucleotide, wherein at least a portion ofthe third oligonucleotide is completely complementary to the firstportion of the second target nucleic acid; generating a first cleavagestructure wherein at least said portion of the first oligonucleotide isannealed to the first region of the first target nucleic acid andwherein at least the 5′ portion of the second oligonucleotide isannealed to the second region of the first target nucleic acid andwherein cleavage of the first cleavage structure occurs via the cleavageagent thereby cleaving the first oligonucleotide to generate a fourtholigonucleotide, said fourth oligonucleotide comprising a 3′ portion anda 5′ portion, wherein the 5′ portion is completely complementary to thesecond portion of the second target nucleic acid; generating a secondcleavage structure under conditions wherein at least said portion of thethird oligonucleotide is annealed to the first region of the secondtarget nucleic acid and wherein at least the 5′ portion of the fourtholigonucleotide is annealed to the second region of the second targetnucleic acid and wherein cleavage of the second cleavage structureoccurs to generate a cleavage fragment; and detecting the cleavage ofthe second cleavage structure. In some preferred embodiments, the 3′portion of the fourth oligonucleotide comprises a 3′ terminal nucleotidenot complementary to the second target nucleic acid. In someembodiments, the 3′ portion of the third oligonucleotide is covalentlylinked to the second target nucleic acid. In some embodiments, thesecond target nucleic acid further comprises a 5′ region, wherein the 5′region of the second target nucleic acid is the third oligonucleotide.The present invention further provides kits comprising: a cleavageagent; a first oligonucleotide comprising a 5′ portion complementary toa first region of a target nucleic acid; and a second oligonucleotidecomprising a 3′ portion and a 5′ portion, said 5′ portion complementaryto a second region of the target nucleic acid downstream of andcontiguous to the first portion. In some embodiments, the 3′ portion ofthe second oligonucleotide comprises a 3′ terminal nucleotide notcomplementary to the target nucleic acid. In preferred embodiments, the3′ portion of the second oligonucleotide consists of a single nucleotidenot complementary to the target nucleic acid. In some embodiments, thekit further comprises a solid support. For example, in some embodiments,the first and/or second oligonucleotide is attached to said solidsupport. In some embodiments, the kit further comprises a buffersolution. In some preferred embodiments, the buffer solution comprises asource of divalent cations (e.g., Mn²⁺ and/or Mg²⁺ ions). In somespecific embodiments, the kit further comprises a third oligonucleotidecomplementary to a third portion of the target nucleic acid upstream ofthe first portion of the first target nucleic acid. In yet otherembodiments, the kit further comprises a target nucleic acid. In someembodiments, the kit further comprises a second target nucleic acid. Inyet other embodiments, the kit further comprises a third oligonucleotidecomprising a 5′ portion complementary to a first region of the secondtarget nucleic acid. In some specific embodiments, the 3′ portion of thethird oligonucleotide is covalently linked to the second target nucleicacid. In other specific embodiments, the second target nucleic acidfurther comprises a 5′ portion, wherein the 5′ portion of the secondtarget nucleic acid is the third oligonucleotide. In still otherembodiments, the kit further comprises an ARRESTOR molecule (e.g.,ARRESTOR oligonucleotide).

The present invention further provides a composition comprising acleavage structure, the cleavage structure comprising: a) a targetnucleic acid, the target nucleic acid having a first region, a secondregion, a third region and a fourth region, wherein the first region islocated adjacent to and downstream from the second region, the secondregion is located adjacent to and downstream from the third region andthe third region is located adjacent to and downstream from the fourthregion; b) a first oligonucleotide complementary to the fourth region ofthe target nucleic acid; c) a second oligonucleotide having a 5′ portionand a 3′ portion wherein the 5′ portion of the second oligonucleotidecontains a sequence complementary to the second region of the targetnucleic acid and wherein the 3′ portion of the second oligonucleotidecontains a sequence complementary to the third region of the targetnucleic acid; and d) a third oligonucleotide having a 5′ portion and a3′ portion wherein the 5′ portion of the third oligonucleotide containsa sequence complementary to the first region of the target nucleic acidand wherein the 3′ portion of the third oligonucleotide contains asequence complementary to the second region of the target nucleic acid.

The present invention is not limited by the length of the four regionsof the target nucleic acid. In one embodiment, the first region of thetarget nucleic acid has a length of 11 to 50 nucleotides. In anotherembodiment, the second region of the target nucleic acid has a length ofone to three nucleotides. In another embodiment, the third region of thetarget nucleic acid has a length of six to nine nucleotides. In yetanother embodiment, the fourth region of the target nucleic acid has alength of 6 to 50 nucleotides.

The invention is not limited by the nature or composition of the of thefirst, second, third and fourth oligonucleotides; these oligonucleotidesmay comprise DNA, RNA, PNA and combinations thereof as well as comprisemodified nucleotides, universal bases, adducts, etc. Further, one ormore of the first, second, third and the fourth oligonucleotides maycontain a dideoxynucleotide at the 3′ terminus.

In one preferred embodiment, the target nucleic acid is not completelycomplementary to at least one of the first, the second, the third andthe fourth oligonucleotides. In a particularly preferred embodiment, thetarget nucleic acid is not completely complementary to the secondoligonucleotide.

As noted above, the present invention contemplates the use ofstructure-specific nucleases in detection methods. In one embodiment,the present invention provides a method of detecting the presence of atarget nucleic acid molecule by detecting non-target cleavage productscomprising: a) providing: i) a cleavage means, ii) a source of targetnucleic acid, the target nucleic acid having a first region, a secondregion, a third region and a fourth region, wherein the first region islocated adjacent to and downstream from the second region, the secondregion is located adjacent to and downstream from the third region andthe third region is located adjacent to and downstream from the fourthregion; iii) a first oligonucleotide complementary to the fourth regionof the target nucleic acid; iv) a second oligonucleotide having a 5′portion and a 3′ portion wherein the 5′ portion of the secondoligonucleotide contains a sequence complementary to the second regionof the target nucleic acid and wherein the 3′ portion of the secondoligonucleotide contains a sequence complementary to the third region ofthe target nucleic acid; iv) a third oligonucleotide having a 5′ and a3′ portion wherein the 5′ portion of the third oligonucleotide containsa sequence complementary to the first region of the target nucleic acidand wherein the 3′ portion of the third oligonucleotide contains asequence complementary to the second region of the target nucleic acid;b) mixing the cleavage means, the target nucleic acid, the firstoligonucleotide, the second oligonucleotide and the thirdoligonucleotide to create a reaction mixture under reaction conditionssuch that the first oligonucleotide is annealed to the fourth region ofthe target nucleic acid and wherein at least the 3′ portion of thesecond oligonucleotide is annealed to the target nucleic acid andwherein at least the 5′ portion of the third oligonucleotide is annealedto the target nucleic acid so as to create a cleavage structure andwherein cleavage of the cleavage structure occurs to generate non-targetcleavage products, each non-target cleavage product having a 3′-hydroxylgroup; and c) detecting the non-target cleavage products.

The invention is not limited by the nature of the target nucleic acid.In one embodiment, the target nucleic acid comprises single-strandedDNA. In another embodiment, the target nucleic acid comprisesdouble-stranded DNA and prior to step c), the reaction mixture istreated such that the double-stranded DNA is rendered substantiallysingle-stranded. In another embodiment, the target nucleic acidcomprises RNA and the first and second oligonucleotides comprise DNA.

The invention is not limited by the nature of the cleavage means. In oneembodiment, the cleavage means is a structure-specific nuclease;particularly preferred structure-specific nucleases are thermostablestructure-specific nucleases. In one preferred embodiment, thethermostable structure-specific nuclease is encoded by a DNA sequenceselected from the group consisting of SEQ ID NOS:1-3, 9, 10, 12, 21, 30,31, 101, 106, 110, 114, 129, 131, 132, 137, 140, 141, 142, 143, 144,145, 147, 150, 151, 153, 155, 156, 157, 158, 161, 163, 178, 180, and182.

In another preferred embodiment, the thermostable structure-specificnuclease is a nuclease from the FEN-1/RAD2/XPG class of nucleases. Inanother preferred embodiment the thermostable structure specificnuclease is a chimerical nuclease.

In an alternative preferred embodiment, the detection of the non-targetcleavage products comprises electrophoretic separation of the productsof the reaction followed by visualization of the separated non-targetcleavage products.

In another preferred embodiment, one or more of the first, second, andthird oligonucleotides contain a dideoxynucleotide at the 3′ terminus.When dideoxynucleotide-containing oligonucleotides are employed, thedetection of the non-target cleavage products preferably comprises: a)incubating the non-target cleavage products with a template-independentpolymerase and at least one labeled nucleoside triphosphate underconditions such that at least one labeled nucleotide is added to the3′-hydroxyl group of the non-target cleavage products to generatelabeled non-target cleavage products; and b) detecting the presence ofthe labeled non-target cleavage products. The invention is not limitedby the nature of the template-independent polymerase employed; in oneembodiment, the template-independent polymerase is selected from thegroup consisting of terminal deoxynucleotidyl transferase (TdT) and polyA polymerase. When TdT or polyA polymerase are employed in the detectionstep, the second oligonucleotide may contain a 5′ end label, the 5′ endlabel being a different label than the label present upon the labelednucleoside triphosphate. The invention is not limited by the nature ofthe 5′ end label; a wide variety of suitable 5′ end labels are known tothe art and include biotin, fluorescein, tetrachlorofluorescein,hexachlorofluorescein, Cy3 amidite, Cy5 amidite and digoxigenin.

In another embodiment, detecting the non-target cleavage productscomprises: a) incubating the non-target cleavage products with atemplate-independent polymerase and at least one nucleoside triphosphateunder conditions such that at least one nucleotide is added to the3′-hydroxyl group of the non-target cleavage products to generate tailednon-target cleavage products; and b) detecting the presence of thetailed non-target cleavage products. The invention is not limited by thenature of the template-independent polymerase employed; in oneembodiment, the template-independent polymerase is selected from thegroup consisting of terminal deoxynucleotidyl transferase (TdT) and polyA polymerase. When TdT or polyA polymerase are employed in the detectionstep, the second oligonucleotide may contain a 5′ end label. Theinvention is not limited by the nature of the 5′ end label; a widevariety of suitable 5′ end labels are known to the art and includebiotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3amidite, Cy5 amidite and digoxigenin.

In a preferred embodiment, the reaction conditions comprise providing asource of divalent cations; particularly preferred divalent cations areMn²⁺ and Mg²⁺ ions.

The present invention further provides a method of detecting thepresence of a target nucleic acid molecule by detecting non-targetcleavage products comprising: a) providing: i) a cleavage means, ii) asource of target nucleic acid, the target nucleic acid having a firstregion, a second region and a third region, wherein the first region islocated adjacent to and downstream from the second region and whereinthe second region is located adjacent to and downstream from the thirdregion; iii) a first oligonucleotide having a 5′ and a 3′ portionwherein the 5′ portion of the first oligonucleotide contains a sequencecomplementary to the second region of the target nucleic acid andwherein the 3′ portion of the first oligonucleotide contains a sequencecomplementary to the third region of the target nucleic acid; iv) asecond oligonucleotide having a length between eleven to fifteennucleotides and further having a 5′ and a 3′ portion wherein the 5′portion of the second oligonucleotide contains a sequence complementaryto the first region of the target nucleic acid and wherein the 3′portion of the second oligonucleotide contains a sequence complementaryto the second region of the target nucleic acid; b) mixing the cleavagemeans, the target nucleic acid, the first oligonucleotide and the secondoligonucleotide to create a reaction mixture under reaction conditionssuch that at least the 3′ portion of the first oligonucleotide isannealed to the target nucleic acid and wherein at least the 5′ portionof the second oligonucleotide is annealed to the target nucleic acid soas to create a cleavage structure and wherein cleavage of the cleavagestructure occurs to generate non-target cleavage products, eachnon-target cleavage product having a 3′-hydroxyl group; and c) detectingthe non-target cleavage products. In a preferred embodiment the cleavagemeans is a structure-specific nuclease, preferably a thermostablestructure-specific nuclease.

The invention is not limited by the length of the various regions of thetarget nucleic acid. In a preferred embodiment, the second region of thetarget nucleic acid has a length between one to five nucleotides. Inanother preferred embodiment, one or more of the first and the secondoligonucleotides contain a dideoxynucleotide at the 3′ terminus. Whendideoxynucleotide-containing oligonucleotides are employed, thedetection of the non-target cleavage products preferably comprises: a)incubating the non-target cleavage products with a template-independentpolymerase and at least one labeled nucleoside triphosphate underconditions such that at least one labeled nucleotide is added to the3′-hydroxyl group of the non-target cleavage products to generatelabeled non-target cleavage products; and b) detecting the presence ofthe labeled non-target cleavage products. The invention is not limitedby the nature of the template-independent polymerase employed; in oneembodiment, the template-independent polymerase is selected from thegroup consisting of terminal deoxynucleotidyl transferase (TdT) and polyA polymerase. When TdT or polyA polymerase is employed in the detectionstep, the second oligonucleotide may contain a 5′ end label, the 5′ endlabel being a different label than the label present upon the labelednucleoside triphosphate. The invention is not limited by the nature ofthe 5′ end label; a wide variety of suitable 5′ end labels are known tothe art and include biotin, fluorescein, tetrachlorofluorescein,hexachlorofluorescein, Cy3 amidite, Cy5 amidite and digoxigenin.

In another embodiment, detecting the non-target cleavage productscomprises: a) incubating the non-target cleavage products with atemplate-independent polymerase and at least one nucleoside triphosphateunder conditions such that at least one nucleotide is added to the3′-hydroxyl group of the non-target cleavage products to generate tailednon-target cleavage products; and b) detecting the presence of thetailed non-target cleavage products. The invention is not limited by thenature of the template-independent polymerase employed; in oneembodiment, the template-independent polymerase is selected from thegroup consisting of terminal deoxynucleotidyl transferase (TdT) and polyA polymerase. When TdT or polyA polymerases are employed in thedetection step, the second oligonucleotide may contain a 5′ end label.The invention is not limited by the nature of the 5′ end label; a widevariety of suitable 5′ end labels are known to the art and includebiotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3amidite, Cy5 amidite and digoxigenin.

The novel detection methods of the invention may be employed for thedetection of target DNAs and RNAs including, but not limited to, targetDNAs and RNAs comprising wild type and mutant alleles of genes,including genes from humans or other animals that are or may beassociated with disease or cancer. In addition, the methods of theinvention may be used for the detection of and/or identification ofstrains of microorganisms, including bacteria, fungi, protozoa, ciliatesand viruses (and in particular for the detection and identification ofRNA viruses, such as HCV).

The present invention further provides improved enzymatic cleavagemeans. In one embodiment, the present invention provides a thermostablestructure-specific nuclease having an amino acid sequence selected fromthe group consisting of SEQ ID NOS:102, 107, 130, 132, 179, 181, 183,184, 185, 186, 187, and 188. In another embodiment, the nuclease isencoded by a DNA sequence selected from the group consisting of SEQ IDNOS:101, 106, 129 131, 178, 180, and 182.

The present invention also provides a recombinant DNA vector comprisingDNA having a nucleotide sequence encoding a structure-specific nuclease,the nucleotide sequence selected from the group consisting of SEQ IDNOS:101, 106, 129 131, 137, 140, 141, 142, 143, 144, 145, 147, 150, 151,153, 155, 156, 157, 158, 161, 163, 178, 180, and 182. In a preferredembodiment, the invention provides a host cell transformed with arecombinant DNA vector comprising DNA having a nucleotide sequenceencoding a structure-specific nuclease, the nucleotide sequence selectedfrom the group consisting of SEQ ID NOS:101, 106, 129, 131, 178, 180,and 182. The invention is not limited by the nature of the host cellemployed. The art is well aware of expression vectors suitable for theexpression of nucleotide sequences encoding structure-specific nucleasesthat can be expressed in a variety of prokaryotic and eukaryotic hostcells. In a preferred embodiment, the host cell is an Escherichia colicell.

The present invention provides purified FEN-1 endonucleases. In oneembodiment, the present invention provides Pyrococcus woesei FEN-1endonuclease. In a preferred embodiment, the purified Pyrococcus woeseiFEN-1 endonuclease has a molecular weight of about 38.7 kilodaltons (themolecular weight may be conveniently estimated using SDS-PAGE asdescribed in Ex. 28).

The present invention further provides an isolated oligonucleotideencoding a Pyrococcus woesei FEN-1 endonuclease, the oligonucleotidehaving a region capable of hybridizing to an oligonucleotide sequenceselected from the group consisting of SEQ ID NOS:116-119. In a preferredembodiment, the oligonucleotide encoding the purified Pyrococcus woeseiFEN-1 endonuclease is operably linked to a heterologous promoter. Thepresent invention is not limited by the nature of the heterologouspromoter employed; in a preferred embodiment, the heterologous promoteris an inducible promoter (the promoter chosen will depend upon the hostcell chosen for expression as is known in the art). The invention is notlimited by the nature of the inducible promoter employed. Preferredinducible promoters include the —P_(L) promoter, the tac promoter, thetrp promoter and the trc promoter.

In another preferred embodiment, the invention provides a recombinantDNA vector comprising an isolated oligonucleotide encoding a Pyrococcuswoesei (Pwo) FEN-1 endonuclease, the oligonucleotide having a regioncapable of hybridizing to an oligonucleotide sequence selected from thegroup consisting of SEQ ID NOS:116-119. Host cells transformed withthese recombinant vectors are also provided. In a preferred embodiment,the invention provides a host cell transformed with a recombinant DNAvector comprising DNA having a region capable of hybridizing to anoligonucleotide sequence selected from the group consisting of SEQ IDNOS:116-119; these vectors may further comprise a heterologous promoteroperably linked to the Pwo FEN-1-encoding polynucleotides. The inventionis not limited by the nature of the host cell employed. The art is wellaware of expression vectors suitable for the expression of PwoFEN-1-encoding polynucleotides that can be expressed in a variety ofprokaryotic and eukaryotic host cells. In a preferred embodiment, thehost cell is an Escherichia coli cell.

In yet another embodiment, the invention provides an isolatedoligonucleotide comprising a gene encoding a Pyrococcus woesei FEN-1endonuclease having a molecular weight of about 38.7 kilodaltons. Inanother embodiment, the encoding a Pyrococcus woesei FEN-1 endonucleaseis operably linked to a heterologous promoter. The present invention isnot limited by the nature of the heterologous promoter employed; in apreferred embodiment, the heterologous promoter is an inducible promoter(the promoter chosen will depend upon the host cell chosen forexpression as is known in the art). The invention is not limited by thenature of the inducible promoter employed. Preferred inducible promotersinclude the —P_(L) promoter, the tac promoter, the trp promoter and thetrc promoter.

The invention further provides recombinant DNA vectors comprising DNAhaving a nucleotide sequence encoding FEN-1 endonucleases. In onepreferred embodiment, the present invention provides a Pyrococcus woeseiFEN-1 endonuclease having a molecular weight of about 38.7 kilodaltons.Still further, a host cell transformed with a recombinant DNA vectorcomprising DNA having a nucleotide sequence encoding FEN-1 endonuclease.In a preferred embodiment, the host cell is transformed with arecombinant DNA vector comprising DNA having a nucleotide sequenceencoding a Pyrococcus woesei FEN-1 endonuclease having a molecularweight of about 38.7 kilodaltons is provided. The invention is notlimited by the nature of the host cell employed. The art is well awareof expression vectors suitable for the expression of Pwo FEN-1-encodingpolynucleotides which can be expressed in a variety of prokaryotic andeukaryotic host cells. In a preferred embodiment, the host cell is anEscherichia coli cell.

Thus, the present invention provides multiple purified FEN-1endonucleases, both purified native forms of the endonucleases, as wellas recombinant endonucleases. In preferred embodiments, the purifiedFEN-1 endonucleases are obtained from archaebacterial or eubacterialorganisms. In particularly preferred embodiments, the FEN-1endonucleases are obtained from organisms selected from the groupconsisting of Archaeoglobus fulgidus, Methanobacteriumthermoautotrophicum, Sulfolobus solfataricus, Pyrobaculum aerophilum,Thermococcus litoralis, Archaeaglobus veneficus, Archaeaglobusprofundus, Acidianus brierlyi, Acidianus ambivalens, Desulfurococcusamylolyticus, Desulfurococcus mobilis, Pyrodictium brockii, Thermococcusgorgonarius, Thermococcus zilligii, Methanopyrus kandleri, Methanococcusigneus, Pyrococcus horikoshii, and Aeropyrum pernix. In a preferredembodiment, the purified FEN-1 endonucleases have molecular weights ofabout 39 kilodaltons (the molecular weight may be conveniently estimatedusing SDS-PAGE as described in Ex. 28).

The present invention further provides isolated oligonucleotidesencoding Archaeoglobus fulgidus and Methanobacterium thermoautotrophicumFEN-1 endonucleases, the oligonucleotides each having a region capableof hybridizing to at least a portion of an oligonucleotide sequence,wherein the oligonucleotide sequence is selected from the groupconsisting of SEQ ID NOS:170, 171, 172, and 173. In some preferredembodiment, the oligonucleotides encoding the Archaeoglobus fulgidus andMethanobacterium thermoautotrophicum FEN-1 endonucleases are operablylinked to heterologous promoters. However, it is not intended that thepresent invention be limited by the nature of the heterologous promoteremployed. It is contemplated that the promoter chosen will depend uponthe host cell chosen for expression as is known in the art. In somepreferred embodiments, the heterologous promoter is an induciblepromoter. The invention is not limited by the nature of the induciblepromoter employed. Preferred inducible promoters include the —P_(L)promoter, the tac promoter, the trp promoter and the trc promoter.

In another preferred embodiment, the invention provides recombinant DNAvectors comprising isolated oligonucleotides encoding Archaeoglobusfulgidus or Methanobacterium thermoautotrophicum FEN-1 endonucleases,each oligonucleotides having a region capable of hybridizing to at leasta portion of an oligonucleotide sequence, wherein the oligonucleotidesequence is selected from the group consisting of SEQ ID NOS:170, 171,172, and 173. The present invention further provides host cellstransformed with these recombinant vectors. In a preferred embodiment,the invention provides a host cell transformed with a recombinant DNAvector comprising DNA having a region capable of hybridizing to at leasta portion of an oligonucleotide sequence, wherein the oligonucleotidesequence is selected from the group consisting of SEQ ID NOS:170, 171,172 and 173. In some embodiments, these vectors may further comprise aheterologous promoter operably linked to the FEN-1-encodingpolynucleotides. The invention is not limited by the nature of the hostcell employed. The art is well aware of expression vectors suitable forthe expression of FEN-1-encoding polynucleotides which can be expressedin a variety of prokaryotic and eukaryotic host cells. In a preferredembodiment, the host cell is an Escherichia coli cell.

The present invention further provides chimeric structure-specificnucleases. In one embodiment, the present invention provides chimericendonucleases comprising amino acid portions derived from theendonucleases selected from the group of FEN-1, XPG and RAD homologs. Ina preferred embodiment, the chimeric endonucleases comprise amino acidportions derived from the FEN-1 endonucleases selected from the group ofPyrococcus furiosus, Methanococcus jannaschi, Pyrococcus woesei,Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, Sulfolobussolfataricus, Pyrobaculum aerophilum, Thermococcus litoralis,Archaeaglobus veneficus, Archaeaglobus profundus, Acidianus brierlyi,Acidianus ambivalens, Desulfurococcus amylolyticus, Desulfurococcusmobilis, Pyrodictium brockii, Thermococcus gorgonarius, Thermococcuszilligii, Methanopyrus kandleri, Methanococcus igneus, Pyrococcushorikoshii, and Aeropyrum pernix. In a more preferred embodiment, thechimeric FEN-1 endonucleases have molecular weights of about 39kilodaltons (the molecular weight may be conveniently estimated usingSDS-PAGE as described in Ex. 28). In another embodiment, the chimericFEN endoncleases have amino acid sequences selected from the group ofSEQ ID NOs: 418, 426, 432, 436, 440, 444, 450, 452, 470, 472, 474, 476,478, 480, 482, and 484.

The present invention further provides isolated oligonucleotidesencoding chimeric endonucleases. In one embodiment, the oligonucleotidesencoding the chimeric endonucleases comprise nucleic acid sequencesderived from the genes selected from the group of FEN-1, XPG and RADhomologs. In a preferred embodiment the oligonucleotides encoding thechimeric endonucleases comprise nucleic acid sequences derived from thegenes encoding the FEN-1 endonucleases selected from the group ofPyrococcus furiosus, Methanococcus jannaschi, Pyrococcus woesei,Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, Sulfolobussolfataricus, Pyrobaculum aerophilum, Thermococcus litoralis,Archaeaglobus veneficus, Archaeaglobus profundus, Acidianus brierlyi,Acidianus ambivalens, Desulfurococcus amylolyticus, Desulfurococcusmobilis, Pyrodictium brockii, Thermococcus gorgonarius, Thermococcuszilligii, Methanopyrus kandleri, Methanococcus igneus, Pyrococcushorikoshii, and Aeropyrum pernix. In another embodiment, theoligonucleotides encoding the chimeric endonucleases comprise a nucleicacid sequence selected from the group of SEQ ID NOs:417, 425, 431, 435,439, 443, 449, 451, 469, 471, 473, 475, 477, 479, 481, and 483. In aparticularly preferred embodiment, the genes for the chimericendonucleases are operably linked to heterologous promoters. The presentinvention is not limited by the nature of the heterologous promoteremployed. It is contemplated that the promoter chosen will depend uponthe host cell selected for expression, as is known in the art. Inpreferred embodiments, the heterologous promoter is an induciblepromoter. The invention is not limited by the nature of the induciblepromoter employed. Preferred inducible promoter include the —P_(L)promoter, the tac promoter, the trp promoter and the trc promoter.

In another preferred embodiment, the invention provides recombinant DNAvectors comprising isolated oligonucleotides encoding the chimericendonucleases described above. In one embodiment, the recombinant DNAvectors comprise isolated oligonucleotides encoding nucleic acidsequences derived from the genes selected from the group of FEN-1, XPGand RAD homologs. In a preferred embodiment, the recombinant DNA vectorscomprise isolated oligonucleotides encoding the chimeric endonucleasescomprising nucleic acid sequences derived from the genes encoding theFEN-1 endonucleases selected from the group of Pyrococcus furiosus,Methanococcus jannaschi, Pyrococcus woesei, Archaeoglobus fulgidus,Methanobacterium thermoautotrophicum, Sulfolobus solfataricus,Pyrobaculum aerophilum, Thermococcus litoralis, Archaeaglobus veneficus,Archaeaglobus profundus, Acidianus brierlyi, Acidianus ambivalens,Desulfurococcus amylolyticus, Desulfurococcus mobilis, Pyrodictiumbrockii, Thermococcus gorgonarius, Thermococcus zilligii, Methanopyruskandleri, Methanococcus igneus, Pyrococcus horikoshii, and Aeropyrumpernix. In another embodiment, the recombinant DNA vectors comprisenucleic acid sequences selected from the group of SEQ ID NOs: 417, 425,431, 435, 439, 443, 449, 451, 469, 471, 473, 475, 477, 479, 481, and483. These vectors may further comprise a heterologous promoter operablylinked to the chimeric nuclease-encoding polynucleotides.

Structure-specific 5′ nucleases play an important role in DNAreplication and repair uniquely recognizing an overlap flap DNAsubstrate and processing it into a DNA nick. However, in the absence ofa high resolution structure of the enzyme/DNA complex, the mechanismunderlying this recognition and substrate specificity, which is key tothe enzyme's function, remains unclear. Experiments conducted during thedevelopment of the present invention, however, provide athree-dimensional model of the structure-specific 5′ flap endonucleasefrom Pyrococcus furiosus in its complex with DNA. The model is based onthe known X-ray structure of the enzyme and a variety of biochemical andmolecular dynamics data utilized in the form of distance restraintsbetween the enzyme and the DNA. Contacts between the 5′ flapendonuclease and the sugar-phosphate backbone of the overlap flapsubstrate were identified using enzyme activity assays on substrateswith methylphosphonate or 2′-O-methyl substitutions. The enzymefootprint extends 2-4 base pairs upstream and 8-9 base pairs downstreamof the cleavage site, thus covering 10-13 base pairs of duplex DNA. Thefootprint data are consistent with a model in which the substrate isbound in the DNA-binding groove such that the downstream duplexinteracts with the helix-hairpin-helix motif of the enzyme. Moleculardynamics simulations to identify the substrate orientation in this modelare consistent with the results of the enzyme activity assays on themethylphosphonate and 2′-O-methyl-modified substrates. To further refinethe model, 5′ flap endonuclease variants with alanine pointsubstitutions at amino acids expected to contact phosphates in thesubstrate and one deletion mutant were tested in enzyme activity assayson the methylphosphonate-modified substrates. Changes in the enzymefootprint observed for two point mutants, R64A and R94A, and for thedeletion mutant in the enzyme's β_(A)/β_(B) region, were interpreted asbeing the result of specific interactions in the enzyme/DNA complex andwere used as distance restraints in molecular dynamics simulations. Thefinal structure suggests that the substrate's 5′ flap interacts with theenzyme's helical arch and that the helix-hairpin-helix motif interactswith the template strand in the downstream duplex 8 base pairs from thecleavage site. This model suggests specific interactions between the 3′end of the upstream oligonucleotide and the enzyme. The proposedstructure presents the first detailed description of substraterecognition by structure-specific 5′ nucleases.

Host cells transformed with these recombinant vectors are also provided.The invention is not limited by the nature of the host cell employed.The art is well aware of expression vectors suitable for the expressionof FEN-1-encoding polynucleotides which can be expressed in a variety ofprokaryotic and eukaryotic host cells. In a preferred embodiment, thehost cell is an Escherichia coli cell.

The present invention further provides mixtures comprising a firststructure-specific nuclease, wherein the first nuclease consists of apurified FEN-1 endonuclease and a second structure-specific nuclease. Inpreferred embodiments, the second structure-specific nuclease of themixture is selected from the group comprising Pyrococcus woesei FEN-1endonuclease, Pyrococcus furiosus FEN-1, Methanococcus jannaschii FEN-1endonuclease, Methanobacterium thermoautotrophicum FEN-1 endonuclease,Archaeoglobus fulgidus FEN-1, Sulfolobus solfataricus, Pyrobaculumaerophilum, Thermococcus litoralis, Archaeaglobus veneficus,Archaeaglobus profundus, Acidianus brierlyi, Acidianus ambivalens,Desulfurococcus amylolyticus, Desulfurococcus mobilis, Pyrodictiumbrockii, Thermococcus gorgonarius, Thermococcus zilligii, Methanopyruskandleri, Methanococcus igneus, Pyrococcus horikoshii, Aeropyrum pernix,and chimerical FEN-1 endonucleases. In alternative embodiments, thepurified FEN-1 endonuclease of the mixture is selected from the groupconsisting Pyrococcus woesei FEN-1 endonuclease, Pyrococcus furiosusFEN-1 endonuclease, Methanococcus jannaschii FEN-1 endonuclease,Methanobacterium thermoautotrophicum FEN-1 endonuclease, Archaeoglobusfulgidus FEN-1, Sulfolobus solfataricus, Pyrobaculum aerophilum,Thermococcus litoralis, Archaeaglobus veneficus, Archaeaglobusprofundus, Acidianus brierlyi, Acidianus ambivalens, Desulfurococcusamylolyticus, Desulfurococcus mobilis, Pyrodictium brockii, Thermococcusgorgonarius, Thermococcus zilligii, Methanopyrus kandleri, Methanococcusigneus, Pyrococcus horikoshii, Aeropyrum pernix, and chimerical FEN-1endonucleases. In yet other preferred embodiments of the mixture, thesecond nuclease is a 5′ nuclease derived from a thermostable DNApolymerase altered in amino acid sequence such that it exhibits reducedDNA synthetic activity from that of the wild-type DNA polymerase butretains substantially the same 5′ nuclease activity of the wild-type DNApolymerase. In some preferred embodiments of the mixture, the secondnuclease is selected from the group consisting of the Cleavase® BNenzyme, Thermus aquaticus DNA polymerase, Thermus thermophilus DNApolymerase, Escherichia coli Exo III, Saccharomyces cerevisiaeRad1/Rad10 complex.

The present invention also provides methods for treating nucleic acid,comprising: a) providing a purified FEN-1 endonuclease; and a nucleicacid substrate; b) treating the nucleic acid substrate under conditionssuch that the substrate forms one or more cleavage structures; and c)reacting the endonuclease with the cleavage structures so that one ormore cleavage products are produced. In some embodiments, the purifiedFEN-1 endonuclease is selected from the group consisting Pyrococcuswoesei FEN-1 endonuclease, Pyrococcus furiosus FEN-1 endonuclease,Methanococcus jannaschii FEN-1 endonuclease, Methanobacteriumthermoautotrophicum FEN-1 endonuclease, Archaeoglobus fulgidus FEN-1,Sulfolobus solfataricus, Pyrobaculum aerophilum, Thermococcus litoralis,Archaeaglobus veneficus, Archaeaglobus profundus, Acidianus brierlyi,Acidianus ambivalens, Desulfurococcus amylolyticus, Desulfurococcusmobilis, Pyrodictium brockii, Thermococcus gorgonarius, Thermococcuszilligii, Methanopyrus kandleri, Methanococcus igneus, Pyrococcushorikoshii, Aeropyrum pernix, and chimerical FEN-1 endonucleases. Inother embodiments, the method further comprises providing astructure-specific nuclease derived from a thermostable DNA polymerasealtered in amino acid sequence such that it exhibits reduced DNAsynthetic activity from that of the wild-type DNA polymerase but retainssubstantially the same 5′ nuclease activity of the wild-type DNApolymerase.

In alternative embodiments of the methods, a portion of the amino acidsequence of the second nuclease is homologous to a portion of the aminoacid sequence of a thermostable DNA polymerase derived from aeubacterial thermophile of the genus Thermus. In yet other embodiments,the thermophile is selected from the group consisting of Thermusaquaticus, Thermus flavus and Thermus thermophilus. In some alternativeembodiments, the structure-specific nuclease is selected from the groupconsisting of the Cleavase® BN enzyme, Thermus aquaticus DNA polymerase,Thermus thermophilus DNA polymerase, Escherichia coli Exo III,Saccharomyces cerevisiae Rad1/Rad10 complex. In some preferredembodiments, the structure-specific nuclease is the Cleavase® BNnuclease. In yet other embodiments, the nucleic acid of step (a) issubstantially single-stranded. In further embodiments, the nucleic acidis selected from the group consisting of RNA and DNA. In yet furtherembodiments, the nucleic acid of step (a) is double stranded.

In other embodiments of the methods, the treating of step (b) comprises:rendering the double-stranded nucleic acid substantiallysingle-stranded; and exposing the single-stranded nucleic acid toconditions such that the single-stranded nucleic acid has secondarystructure. In some preferred embodiments, the double stranded nucleicacid is rendered substantially single-stranded by the use of increasedtemperature. In alternative preferred embodiments, the method furthercomprises the step of detecting the one or more cleavage products.

The present invention also provides methods for treating nucleic acid,comprising: a) providing: a first structure-specific nuclease consistingof a purified FEN-1 endonuclease in a solution containing manganese; anda nucleic acid substrate; b) treating the nucleic acid substrate withincreased temperature such that the substrate is substantiallysingle-stranded; c) reducing the temperature under conditions such thatthe single-stranded substrate forms one or more cleavage structures; d)reacting the cleavage means with the cleavage structures so that one ormore cleavage products are produced; and e) detecting the one or morecleavage products. In some embodiments of the methods, the purifiedFEN-1 endonuclease is selected from the group consisting Pyrococcuswoesei FEN-1 endonuclease, Pyrococcus furiosus FEN-1 endonuclease,Methanococcus jannaschii FEN-1 endonuclease, Methanobacteriumthermoautotrophicum FEN-1 endonuclease, Archaeoglobus fulgidus FEN-1,Sulfolobus solfataricus, Pyrobaculum aerophilum, Thermococcus litoralis,Archaeaglobus veneficus, Archaeaglobus profundus, Acidianus brierlyi,Acidianus ambivalens, Desulfurococcus amylolyticus, Desulfurococcusmobilis, Pyrodictium brockii, Thermococcus gorgonarius, Thermococcuszilligii, Methanopyrus kandleri, Methanococcus igneus, Pyrococcushorikoshii, Aeropyrum pernix, and chimerical FEN-1 endonucleases. Inalternative embodiments, the methods further comprise providing a secondstructure-specific nuclease. In some preferred embodiments, the secondnuclease is selected from the group consisting of the Cleavase® BNenzyme, Thermus aquaticus DNA polymerase, Thermus thermophilus DNApolymerase, Escherichia coli Exo III, and the Saccharomyces cerevisiaeRad1/Rad10 complex. In yet other preferred embodiments, the secondnuclease is a 5′ nuclease derived from a thermostable DNA polymerasealtered in amino acid sequence such that it exhibits reduced DNAsynthetic activity from that of the wild-type DNA polymerase but retainssubstantially the same 5′ nuclease activity of the wild-type DNApolymerase. In yet other embodiments, the nucleic acid is selected fromthe group consisting of RNA and DNA. In further embodiments, the nucleicacid of step (a) is double stranded.

The present invention also provides nucleic acid treatment kits,comprising: a) a composition comprising at least one purified FEN-1endonuclease; and b) a solution containing manganese. In someembodiments of the kits, the purified FEN-1 endonuclease is selectedfrom the group consisting Pyrococcus woesei FEN-1 endonuclease,Pyrococcus furiosus FEN-1 endonuclease, Methanococcus jannaschii FEN-1endonuclease, Methanobacterium thermoautotrophicum FEN-1 endonuclease,Archaeoglobus fulgidus FEN-1, Sulfolobus solfataricus, Pyrobaculumaerophilum, Thermococcus litoralis, Archaeaglobus veneficus,Archaeaglobus profundus, Acidianus brierlyi, Acidianus ambivalens,Desulfurococcus amylolyticus, Desulfurococcus mobilis, Pyrodictiumbrockii, Thermococcus gorgonarius, Thermococcus zilligii, Methanopyruskandleri, Methanococcus igneus, Pyrococcus horikoshii, Aeropyrum pernix,and chimerical FEN-1 endonucleases. In other embodiments, the kitsfurther comprise at least one second structure-specific nuclease. Insome preferred embodiments, the second nuclease is a 5′ nuclease derivedfrom a thermostable DNA polymerase altered in amino acid sequence suchthat it exhibits reduced DNA synthetic activity from that of thewild-type DNA polymerase but retains substantially the same 5′ nucleaseactivity of the wild-type DNA polymerase. In yet other embodiments ofthe kits, the portion of the amino acid sequence of the second nucleaseis homologous to a portion of the amino acid sequence of a thermostableDNA polymerase derived from a eubacterial thermophile of the genusThermus. In further embodiments, the thermophile is selected from thegroup consisting of Thermus aquaticus, Thermus flavus and Thermusthermophilus. In yet other preferred embodiments, the kits furthercomprise reagents for detecting the cleavage products.

The present invention further provides any of the compositions,mixtures, methods, and kits described herein, used in conjunction withendonucleases comprising Sulfolobus solfataricus, Pyrobaculumaerophilum, Thermococcus litoralis, Archaeaglobus veneficus,Archaeaglobus profundus, Acidianus brierlyi, Acidianus ambivalens,Desulfurococcus amylolyticus, Desulfurococcus mobilis, Pyrodictiumbrockii, Thermococcus gorgonarius, Thermococcus zilligii, Methanopyruskandleri, Methanococcus igneus, Pyrococcus horikoshii, and Aeropyrumpernix endonucleases. These include compositions comprising purifiedFEN-1 endonucleases from the organisms (including specific endonucleasesdescribed by sequences provided herein, as well as, variants andhomologues), kits comprising these compositions, composition comprisingchimerical endonucleases comprising at least a portion of theendonucleases from these organisms, kits comprising such compositions,compositions comprising nucleic acids encoding the endonucleases fromthese organisms (including vectors and host cells), kits comprising suchcompositions, antibodies generated to the endonucleases, mixturescomprising endonucleases from these organisms, methods of using theendonuclease in cleavage assays (e.g., invasive cleavage assays, CFLP,etc.), and kits containing components useful for such methods. Exampledescribing the generation, structure, use, and characterization of theseendonucleases are provided in Examples 62-67.

The present invention also provides methods for improving the methodsand enzymes disclosed herein. For example, the present inventionprovides methods of improving enzymes for any intended purpose (e.g.,use in cleavage reactions, amplification reactions, binding reactions,or any other use) comprising the step of providing an enzyme disclosedherein and modifying the enzyme (e.g., altering the amino acid sequence,adding or subtracting sequence, adding post-translational modifications,adding any other component whether biological or not, or any othermodification). Likewise, the present invention provides methods forimproving the methods disclosed herein comprising, conducting the methodsteps with one or more changes (e.g., change in a composition providedin the method, change in the order of the steps, or addition orsubtraction of steps).

The improved performance in a detection assay may arise from any one of,or a combination of several improved features. For example, in oneembodiment, the enzyme of the present invention may have an improvedrate of cleavage (kcat) on a specific targeted structure, such that alarger amount of a cleavage product may be produced in a given timespan. In another embodiment, the enzyme of the present invention mayhave a reduced activity or rate in the cleavage of inappropriate ornon-specific structures. For example, in certain embodiments of thepresent invention, one aspect of improvement is that the differentialbetween the detectable amount of cleavage of a specific structure andthe detectable amount of cleavage of any alternative structures isincreased. As such, it is within the scope of the present invention toprovide an enzyme having a reduced rate of cleavage of a specific targetstructure compared to the rate of the native enzyme, and having afurther reduced rate of cleavage of any alternative structures, suchthat the differential between the detectable amount of cleavage of thespecific structure and the detectable amount of cleavage of anyalternative structures is increased. However, the present invention isnot limited to enzymes that have an improved differential.

The present invention contemplates structure-specific nucleases from avariety of sources, including, but not limited to, mesophilic,psychrophilic, thermophilic, and hyperthermophilic organisms. Thepreferred structure-specific nucleases are thermostable. Thermostablestructure-specific nucleases are contemplated as particularly useful inthat they allow the INVADER assay (Third Wave Technologies, described inU.S. Pat. Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543,6,348,314, and 6,458,535, WO 97/27214 WO 98/42873, Lyamichev et al.,Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000),each of which is herein incorporated by reference in its entirety forall purposes) to be operated near the melting temperature (T_(m)) of thedownstream probe oligonucleotide, so that cleaved and uncleaved probesmay cycle on and off the target during the course of the reaction. Inone embodiment, the thermostable structure-specific enzymes arethermostable 5′ nucleases that are selected from the group comprisingaltered polymerases derived from the native polymerases of Thermusspecies, including, but not limited to, Thermus aquaticus, Thermusflavus, Thermus thermophilus, Thermus filiformus, and Thermusscotoductus. However, the invention is not limited to the use ofthermostable 5′ nucleases. For example, certain embodiments of thepresent invention utilize short oligonucleotide probes that may cycle onand off of the target at low temperatures, allowing the use ofnon-thermostable enzymes.

In some preferred embodiments, the present invention provides acomposition comprising an enzyme, wherein the enzyme comprises aheterologous functional domain, wherein the heterologous functionaldomain provides altered (e.g., improved) functionality in a nucleic acidcleavage assay. The present invention is not limited by the nature ofthe nucleic acid cleavage assay. For example, nucleic acid cleavageassays include any assay in which a nucleic acid is cleaved, directly orindirectly, in the presence of the enzyme. In certain preferredembodiments, the nucleic acid cleavage assay is an invasive cleavageassay. In particularly preferred embodiments, the cleavage assayutilizes a cleavage structure having at least one RNA component. Inanother particularly preferred embodiment, the cleavage assay utilizes acleavage structure having at least one RNA component, wherein a DNAmember of the cleavage structure is cleaved.

The present invention is not limited by the nature of the alteredfunctionality provided by the heterologous functional domain.Illustrative examples of alterations include, but are not limited to,enzymes where the heterologous functional domain comprises an amino acidsequence (e.g., one or more amino acids) that provides an improvednuclease activity, an improved substrate binding activity and/orimproved background specificity in a nucleic acid cleavage assay.

The present invention is not limited by the nature of the heterologousfunctional domain. For example, in some embodiments, the heterologousfunctional domain comprises two or more amino acids from a polymerasedomain of a polymerase (e.g., introduced into the enzyme by insertion ofa chimerical functional domain or created by mutation). In certainpreferred embodiment, at least one of the two or more amino acids isfrom a palm or thumb region of the polymerase domain. The presentinvention is not limited by the identity of the polymerase from whichthe two or more amino acids are selected. In certain preferredembodiments, the polymerase comprises Thermus thermophilus polymerase.In particularly preferred embodiments, the two or more amino acids arefrom amino acids 300-650 of SEQ ID NO:267.

The novel enzymes of the invention may be employed for the detection oftarget DNAs and RNAs including, but not limited to, target DNAs and RNAscomprising wild type and mutant alleles of genes, including, but notlimited to, genes from humans, other animal, or plants that are or maybe associated with disease or other conditions. In addition, the enzymesof the invention may be used for the detection of and/or identificationof strains of microorganisms, including bacteria, fungi, protozoa,ciliates and viruses (and in particular for the detection andidentification of viruses having RNA genomes, such as the Hepatitis Cand Human Immunodeficiency viruses). For example, the present inventionprovides methods for cleaving a nucleic acid comprising providing: anenzyme of the present invention and a substrate nucleic acid; andexposing the substrate nucleic acid to the enzyme (e.g., to produce acleavage product that may be detected).

In some embodiments the present invention provides a method fordetecting a target sequence, comprising the steps of forming a cleavagestructure on the target sequence that is cleavable by a FEN-1endonuclease and cleaving said cleavage structure with a cleavage agentunder conditions such that at least a 5′ nucleotide is removed from anucleic acid molecule contained in said cleavage structure. In someembodiments, the target sequence comprises a secondary structure that isspanned by at least one nucleic acid molecule contained in said cleavagestructure.

In some embodiments, the cleavage structure comprises a non-nucleotideconstituent. In some preferred embodiments, the non-nucleotideconstituent is positioned in said cleavage structure to permit cleavageof said cleavage structure, such that, if said non-nucleotideconstituent was not present in said position, said cleavage structurewould not be cleaved by said cleavage agent.

In some embodiments, the present invention provides a method fordetecting the presence of a target nucleic acid molecule by detectingnon-target cleavage products, comprising providing: a FEN nucleasecomprising a Y33 equivalent residue; a source of target nucleic acid,said target nucleic acid comprising a first region and a second region,said second region downstream of and contiguous to said first region; afirst oligonucleotide, wherein a first portion of said firstoligonucleotide comprises at least one nucleotide analog and whereinsaid first portion is completely complementary to said first portion ofsaid first target nucleic acid, and; an aromatic moiety, combining saidFEN nuclease, said target nucleic acid, said first oligonucleotide, andsaid aromatic moiety under reaction conditions to form a cleavagecomplex, wherein said first portion of said first oligonucleotide isannealed to said first region of said target nucleic acid to form aduplex, wherein said FEN nuclease recognizes said duplex, and whereinsaid aromatic moiety interacts with said Y33 equivalent residue of saidFEN nuclease, such that cleavage of said complex occurs to generate anon-target cleavage product. Some preferred embodiments further comprisedetecting the cleavage of said cleavage complex.

In some embodiments, the interaction of the aromatic moiety with saidY33 equivalent residue of said FEN nuclease comprises a stackinginteraction. In some embodiments, the aromatic moiety is a nucleotide.In other embodiments, the aromatic moiety is a nucleotide analog.

In some embodiments, a second oligonucleotide is provided, said secondoligonucleotide comprising a 3′ portion and a 5′ portion, wherein said5′ portion is completely complementary to said second region of saidtarget nucleic acid. In some preferred embodiments, the 3′ portion ofthe second oligonucleotide comprises the aromatic moiety.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides such asan oligonucleotide or a target nucleic acid) related by the base-pairingrules. For example, for the sequence “5′-A-G-T-3′,” is complementary tothe sequence “3′-T-C-A-5′.” Complementarity may be “partial,” in whichonly some of the nucleic acids' bases are matched according to the basepairing rules. Or, there may be “complete” or “total” complementaritybetween the nucleic acids. The degree of complementarity between nucleicacid strands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids. Either term may also beused in reference to individual nucleotides, especially within thecontext of polynucleotides. For example, a particular nucleotide withinan oligonucleotide may be noted for its complementarity, or lackthereof, to a nucleotide within another nucleic acid strand, in contrastor comparison to the complementarity between the rest of theoligonucleotide and the nucleic acid strand.

The term “homology” and “homologous” refers to a degree of identity.There may be partial homology or complete homology. A partiallyhomologous sequence is one that is less than 100% identical to anothersequence.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is influenced by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, and the T_(m) of the formed hybrid. “Hybridization” methodsinvolve the annealing of one nucleic acid to another, complementarynucleic acid, i.e., a nucleic acid having a complementary nucleotidesequence. The ability of two polymers of nucleic acid containingcomplementary sequences to find each other and anneal through basepairing interaction is a well-recognized phenomenon. The initialobservations of the “hybridization” process by Marmur and Lane, Proc.Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad.Sci. USA 46:461 (1960) have been followed by the refinement of thisprocess into an essential tool of modern biology.

With regard to complementarity, it is important for some diagnosticapplications to determine whether the hybridization represents completeor partial complementarity. For example, where it is desired to detectsimply the presence or absence of pathogen DNA (such as from a virus,bacterium, fungi, mycoplasma, protozoan) it is only important that thehybridization method ensures hybridization when the relevant sequence ispresent; conditions can be selected where both partially complementaryprobes and completely complementary probes will hybridize. Otherdiagnostic applications, however, may require that the hybridizationmethod distinguish between partial and complete complementarity. It maybe of interest to detect genetic polymorphisms. For example, humanhemoglobin is composed, in part, of four polypeptide chains. Two ofthese chains are identical chains of 141 amino acids (alpha chains) andtwo of these chains are identical chains of 146 amino acids (betachains). The gene encoding the beta chain is known to exhibitpolymorphism. The normal allele encodes a beta chain having glutamicacid at the sixth position. The mutant allele encodes a beta chainhaving valine at the sixth position. This difference in amino acids hasa profound (most profound when the individual is homozygous for themutant allele) physiological impact known clinically as sickle cellanemia. It is well known that the genetic basis of the amino acid changeinvolves a single base difference between the normal allele DNA sequenceand the mutant allele DNA sequence.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” Certain bases not commonly found innatural nucleic acids may be included in the nucleic acids of thepresent invention and include, for example, inosine and 7-deazaguanine.Complementarity need not be perfect; stable duplexes may containmismatched base pairs or unmatched bases. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. Several equations for calculating theT_(m) of nucleic acids are well known in the art. As indicated bystandard references, a simple estimate of the T_(m) value may becalculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl (see e.g., Anderson and Young,Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985).Other references (e.g., Allawi, H. T. & SantaLucia, J., Jr.Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry36, 10581-94 (1997) include more sophisticated computations which takestructural and environmental, as well as sequence characteristics intoaccount for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. With“high stringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “weak” or “low”stringency are often required when it is desired that nucleic acidswhich are not completely complementary to one another be hybridized orannealed together.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42 C in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42 C when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42 C in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42 C when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42 C in a solution consisting of 5×SSPE (43.8 g/l NaCl,6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH),0.1% SDS, 5× Denhardt's reagent [50× Denhardt's contains per 500 ml: 5 gFicoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 g/ldenatured salmon sperm DNA followed by washing in a solution comprising5×SSPE, 0.1% SDS at 42 C when a probe of about 500 nucleotides in lengthis employed.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of an RNA having anon-coding function (e.g., a ribosomal or transfer RNA), a polypeptideor a precursor. The RNA or polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence so long as thedesired activity or function is retained.

The term “wild-type” refers to a gene or a gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified,” “mutant,” or “polymorphic” refers to a gene or gene productwhich displays modifications in sequence and or functional properties(i.e., altered characteristics) when compared to the wild-type gene orgene product. It is noted that naturally-occurring mutants can beisolated; these are identified by the fact that they have alteredcharacteristics when compared to the wild-type gene or gene product.

The term “recombinant DNA vector” as used herein refers to DNA sequencescontaining a desired heterologous sequence. For example, although theterm is not limited to the use of expressed sequences or sequences thatencode an expression product, in some embodiments, the heterologoussequence is a coding sequence and appropriate DNA sequences necessaryfor either the replication of the coding sequence in a host organism, orthe expression of the operably linked coding sequence in a particularhost organism. DNA sequences necessary for expression in prokaryotesinclude a promoter, optionally an operator sequence, a ribosome bindingsite and possibly other sequences. Eukaryotic cells are known to utilizepromoters, polyadenlyation signals and enhancers.

The term “LTR” as used herein refers to the long terminal repeat foundat each end of a provirus (i.e., the integrated form of a retrovirus).The LTR contains numerous regulatory signals including transcriptionalcontrol elements, polyadenylation signals and sequences needed forreplication and integration of the viral genome. The viral LTR isdivided into three regions called U3, R and U5.

The U3 region contains the enhancer and promoter elements. The U5 regioncontains the polyadenylation signals. The R (repeat) region separatesthe U3 and U5 regions and transcribed sequences of the R region appearat both the 5′ and 3′ ends of the viral RNA.

The term “oligonucleotide” as used herein is defined as a moleculecomprising two or more deoxyribonucleotides or ribonucleotides,preferably at least 5 nucleotides, more preferably at least about 10-15nucleotides and more preferably at least about 15 to 30 nucleotides. Theexact size will depend on many factors, which in turn depend on theultimate function or use of the oligonucleotide. The oligonucleotide maybe generated in any manner, including chemical synthesis, DNAreplication, reverse transcription, PCR, or a combination thereof.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points towards the 5′ end of the other,the former may be called the “upstream” oligonucleotide and the latterthe “downstream” oligonucleotide. Similarly, when two overlappingoligonucleotides are hybridized to the same linear complementary nucleicacid sequence, with the first oligonucleotide positioned such that its5′ end is upstream of the 5′ end of the second oligonucleotide, and the3′ end of the first oligonucleotide is upstream of the 3′ end of thesecond oligonucleotide, the first oligonucleotide may be called the“upstream” oligonucleotide and the second oligonucleotide may be calledthe “downstream” oligonucleotide.

The term “primer” refers to an oligonucleotide that is capable of actingas a point of initiation of synthesis when placed under conditions inwhich primer extension is initiated. An oligonucleotide “primer” mayoccur naturally, as in a purified restriction digest or may be producedsynthetically.

A primer is selected to be “substantially” complementary to a strand ofspecific sequence of the template. A primer must be sufficientlycomplementary to hybridize with a template strand for primer elongationto occur. A primer sequence need not reflect the exact sequence of thetemplate. For example, a non-complementary nucleotide fragment may beattached to the 5′ end of the primer, with the remainder of the primersequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

The term “label” as used herein refers to any atom or molecule that canbe used to provide a detectable (preferably quantifiable) signal, andthat can be attached to a nucleic acid or protein. Labels may providesignals detectable by fluorescence, radioactivity, colorimetry,gravimetry, X-ray diffraction or absorption, magnetism, enzymaticactivity, and the like. A label may be a charged moiety (positive ornegative charge) or alternatively, may be charge neutral. Labels caninclude or consist of nucleic acid or protein sequence, so long as thesequence comprising the label is detectable.

The term “cleavage structure” as used herein, refers to a structure thatis formed by the interaction of at least one probe oligonucleotide and atarget nucleic acid, forming a structure comprising a duplex, theresulting structure being cleavable by a cleavage means, including butnot limited to an enzyme. The cleavage structure is a substrate forspecific cleavage by the cleavage means in contrast to a nucleic acidmolecule that is a substrate for non-specific cleavage by agents such asphosphodiesterases that cleave nucleic acid molecules without regard tosecondary structure (i.e., no formation of a duplexed structure isrequired).

The term “folded cleavage structure” as used herein, refers to a regionof a single-stranded nucleic acid substrate containing secondarystructure, the region being cleavable by an enzymatic cleavage means.The cleavage structure is a substrate for specific cleavage by thecleavage means in contrast to a nucleic acid molecule that is asubstrate for non-specific cleavage by agents such as phosphodiesteraseswhich cleave nucleic acid molecules without regard to secondarystructure (i.e., no folding of the substrate is required).

As used herein, the term “folded target” refers to a nucleic acid strandthat contains at least one region of secondary structure (i.e., at leastone double stranded region and at least one single-stranded regionwithin a single strand of the nucleic acid). A folded target maycomprise regions of tertiary structure in addition to regions ofsecondary structure.

The term “cleavage means” or “cleavage agent” as used herein refers toany means that is capable of cleaving a cleavage structure, includingbut not limited to enzymes. The cleavage means may include native DNAPshaving 5′ nuclease activity (e.g., Taq DNA polymerase, E. coli DNApolymerase I) and, more specifically, modified DNAPs having 5′ nucleasebut lacking synthetic activity. “Structure-specific nucleases” or“structure-specific enzymes” are enzymes that recognize specificsecondary structures in a nucleic molecule and cleave these structures.The cleavage means of the invention cleave a nucleic acid molecule inresponse to the formation of cleavage structures; it is not necessarythat the cleavage means cleave the cleavage structure at any particularlocation within the cleavage structure.

The cleavage means is not restricted to enzymes having solely 5′nuclease activity. The cleavage means may include nuclease activityprovided from a variety of sources including the Cleavase enzymes, theFEN-1 endonucleases (including RAD2 and XPG proteins), Taq DNApolymerase and E. coli DNA polymerase I.

The term “thermostable” when used in reference to an enzyme, such as a5′ nuclease, indicates that the enzyme is functional or active (i.e.,can perform catalysis) at an elevated temperature, i.e., at about 55° C.or higher.

The term “cleavage products” as used herein, refers-to productsgenerated by the reaction of a cleavage means with a cleavage structure(i.e., the treatment of a cleavage structure with a cleavage means).

The term “target nucleic acid” refers to a nucleic acid moleculecontaining a sequence that has at least partial complementarity with atleast a probe oligonucleotide and may also have at least partialcomplementarity with an INVADER oligonucleotide. The target nucleic acidmay comprise single- or double-stranded DNA or RNA.

The term “probe oligonucleotide” refers to an oligonucleotide thatinteracts with a target nucleic acid to form a cleavage structure in thepresence or absence of an INVADER oligonucleotide. When annealed to thetarget nucleic acid, the probe oligonucleotide and target form acleavage structure and cleavage occurs within the probe oligonucleotide.

The term “non-target cleavage product” refers to a product of a cleavagereaction that is not derived from the target nucleic acid. As discussedabove, in the methods of the present invention, cleavage of the cleavagestructure generally occurs within the probe oligonucleotide. Thefragments of the probe oligonucleotide generated by this target nucleicacid-dependent cleavage are “non-target cleavage products.”

The term “INVADER oligonucleotide” refers to an oligonucleotide thathybridizes to a target nucleic acid at a location near the region ofhybridization between a probe and the target nucleic acid, wherein theINVADER oligonucleotide comprises a portion (e.g., a chemical moiety, ornucleotide—whether complementary to that target or not) that overlapswith the region of hybridization between the probe and target. In someembodiments, the INVADER oligonucleotide contains sequences at its 3′end that are substantially the same as sequences located at the 5′ endof a probe oligonucleotide.

The term “substantially single-stranded” when used in reference to anucleic acid substrate means that the substrate molecule existsprimarily as a single strand of nucleic acid in contrast to adouble-stranded substrate which exists as two strands of nucleic acidwhich are held together by inter-strand base pairing interactions.

The term “sequence variation” as used herein refers to differences innucleic acid sequence between two nucleic acids. For example, awild-type structural gene and a mutant form of this wild-type structuralgene may vary in sequence by the presence of single base substitutionsand/or deletions or insertions of one or more nucleotides. These twoforms of the structural gene are said to vary in sequence from oneanother. A second mutant form of the structural gene may exist. Thissecond mutant form is said to vary in sequence from both the wild-typegene and the first mutant form of the gene.

The term “liberating” as used herein refers to the release of a nucleicacid fragment from a larger nucleic acid fragment, such as anoligonucleotide, by the action of, for example, a 5′ nuclease such thatthe released fragment is no longer covalently attached to the remainderof the oligonucleotide.

The term “K_(m)” as used herein refers to the Michaelis-Menten constantfor an enzyme and is defined as the concentration of the specificsubstrate at which a given enzyme yields one-half its maximum velocityin an enzyme catalyzed reaction.

The term “nucleotide analog” as used herein refers to modified ornon-naturally occurring nucleotides including but not limited to analogsthat have altered stacking interactions such as 7-deaza purines (i.e.,7-deaza-dATP and 7-deaza-dGTP); base analogs with alternative hydrogenbonding configurations (e.g., such as Iso-C and Iso-G and othernon-standard base pairs described in U.S. Pat. No. 6,001,983 to S.Benner); non-hydrogen bonding analogs (e.g., non-polar, aromaticnucleoside analogs such as 2,4-difluorotoluene, described by B. A.Schweitzer and E. T. Kool, J. Org. Chem., 1994, 59, 7238-7242, B. A.Schweitzer and E. T. Kool, J. Am. Chem. Soc., 1995, 117, 1863-1872);“universal” bases such as 5-nitroindole and 3-nitropyrrole; anduniversal purines and pyrimidines (such as “K” and “P” nucleotides,respectively; P. Kong, et al., Nucleic Acids Res., 1989, 17,10373-10383, P. Kong et al., Nucleic Acids Res., 1992, 20, 5149-5152).Nucleotide analogs comprise modified forms of deoxyribonucleotides aswell as ribonucleotides.

The term “polymorphic locus” is a locus present in a population thatshows variation between members of the population (e.g., the most commonallele has a frequency of less than 0.95). In contrast, a “monomorphiclocus” is a genetic locus at little or no variations seen betweenmembers of the population (generally taken to be a locus at which themost common allele exceeds a frequency of 0.95 in the gene pool of thepopulation).

The term “microorganism” as used herein means an organism too small tobe observed with the unaided eye and includes, but is not limited tobacteria, virus, protozoans, fungi, and ciliates.

The term “microbial gene sequences” refers to gene sequences derivedfrom a microorganism.

The term “bacteria” refers to any bacterial species includingeubacterial and archaebacterial species.

The term “virus” refers to obligate, ultramicroscopic, intracellularparasites incapable of autonomous replication (i.e., replicationrequires the use of the host cell's machinery).

The term “multi-drug resistant” or multiple-drug resistant” refers to amicroorganism which is resistant to more than one of the antibiotics orantimicrobial agents used in the treatment of said microorganism.

The term “sample” in the present specification and claims is used in itsbroadest sense. On the one hand it is meant to include a specimen orculture (e.g., microbiological cultures). On the other hand, it is meantto include both biological and environmental samples. A sample mayinclude a specimen of synthetic origin.

Biological samples may be animal, including human, fluid, solid (e.g.,stool) or tissue, as well as liquid and solid food and feed products andingredients such as dairy items, vegetables, meat and meat by-products,and waste. Biological samples may be obtained from all of the variousfamilies of domestic animals, as well as feral or wild animals,including, but not limited to, such animals as ungulates, bear, fish,lagamorphs, rodents, etc.

Environmental samples include environmental material such as surfacematter, soil, water and industrial samples, as well as samples obtainedfrom food and dairy processing instruments, apparatus, equipment,utensils, disposable and non-disposable items. These examples are not tobe construed as limiting the sample types applicable to the presentinvention.

The term “source of target nucleic acid” refers to any sample thatcontains nucleic acids (RNA or DNA). Particularly preferred sources oftarget nucleic acids are biological samples including, but not limitedto blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph,sputum and semen.

An oligonucleotide is said to be present in “excess” relative to anotheroligonucleotide (or target nucleic acid sequence) if thatoligonucleotide is present at a higher molar concentration that theother oligonucleotide (or target nucleic acid sequence). When anoligonucleotide such as a probe oligonucleotide is present in a cleavagereaction in excess relative to the concentration of the complementarytarget nucleic acid sequence, the reaction may be used to indicate theamount of the target nucleic acid present. Typically, when present inexcess, the probe oligonucleotide will be present at least a 100-foldmolar excess; typically at least 1 pmole of each probe oligonucleotidewould be used when the target nucleic acid sequence was present at about10 fmoles or less.

A sample “suspected of containing” a first and a second target nucleicacid may contain either, both or neither target nucleic acid molecule.

The term “charge-balanced” oligonucleotide refers to an oligonucleotide(the input oligonucleotide in a reaction) that has been modified suchthat the modified oligonucleotide bears a charge, such that when themodified oligonucleotide is either cleaved (i.e., shortened) orelongated, a resulting product bears a charge different from the inputoligonucleotide (the “charge-unbalanced” oligonucleotide) therebypermitting separation of the input and reacted oligonucleotides on thebasis of charge. The term “charge-balanced” does not imply that themodified or balanced oligonucleotide has a net neutral charge (althoughthis can be the case). Charge-balancing refers to the design andmodification of an oligonucleotide such that a specific reaction productgenerated from this input oligonucleotide can be separated on the basisof charge from the input oligonucleotide.

For example, in an INVADER oligonucleotide-directed cleavage assay inwhich the probe oligonucleotide bears the sequence: 5′TTCTTTTCACCAGCGAGACGGG 3′ (i.e., SEQ ID NO:61 without the modifiedbases) and cleavage of the probe occurs between the second and thirdresidues, one possible charge-balanced version of this oligonucleotidewould be: 5′ Cy3-AminoT-Amino-TCTTTTCACCAGCGAGAC GGG 3′. This modifiedoligonucleotide bears a net negative charge. After cleavage, thefollowing oligonucleotides are generated: 5′ Cy3-AminoT-Amino-T 3′ and5′ CTTTTCACCAGCGAGACGGG 3′ (residues 3-22of SEQ ID NO:61). 5′Cy3-AminoT-Amino-T 3′ bears a detectable moiety (the positively-chargedCy3 dye) and two amino-modified bases. The amino-modified bases and theCy3 dye contribute positive charges in excess of the negative chargescontributed by the phosphate groups and thus the 5′ Cy3-AminoT-Amino-T3′ oligonucleotide has a net positive charge. The other, longer cleavagefragment, like the input probe, bears a net negative charge. Because the5′ Cy3-AminoT-Amino-T 3′ fragment is separable on the basis of chargefrom the input probe (the charge-balanced oligonucleotide), it isreferred to as a charge-unbalanced oligonucleotide. The longer cleavageproduct cannot be separated on the basis of charge from the inputoligonucleotide as both oligonucleotides bear a net negative charge;thus, the longer cleavage product is not a charge-unbalancedoligonucleotide.

The term “net neutral charge” when used in reference to anoligonucleotide, including modified oligonucleotides, indicates that thesum of the charges present (i.e., R—NH3+ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction or separation conditions is essentially zero.An oligonucleotide having a net neutral charge would not migrate in anelectrical field.

The term “net positive charge” when used in reference to anoligonucleotide, including modified oligonucleotides, indicates that thesum of the charges present (i.e., R—NH3+ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction conditions is +1 or greater. Anoligonucleotide having a net positive charge would migrate toward thenegative electrode in an electrical field.

The term “net negative charge” when used in reference to anoligonucleotide, including modified oligonucleotides, indicates that thesum of the charges present (i.e., R—-NH3+ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction conditions is −1 or lower. An oligonucleotidehaving a net negative charge would migrate toward the positive electrodein an electrical field.

The term “polymerization means” or “polymerization agent” refers to anyagent capable of facilitating the addition of nucleoside triphosphatesto an oligonucleotide. Preferred polymerization means comprise DNA andRNA polymerases.

The term “ligation means” or “ligation agent” refers to any agentcapable of facilitating the ligation (i.e., the formation of aphosphodiester bond between a 3′-OH and a 5′ P located at the termini oftwo strands of nucleic acid). Preferred ligation means comprise DNAligases and RNA ligases.

The term “reactant” is used herein in its broadest sense. The reactantcan comprise, for example, an enzymatic reactant, a chemical reactant orlight (e.g., ultraviolet light, particularly short wavelengthultraviolet light is known to break oligonucleotide chains). Any agentcapable of reacting with an oligonucleotide to either shorten (i.e.,cleave) or elongate the oligonucleotide is encompassed within the term“reactant.”

The term “adduct” is used herein in its broadest sense to indicate anycompound or element that can be added to an oligonucleotide. An adductmay be charged (positively or negatively) or may be charge-neutral. Anadduct may be added to the oligonucleotide via covalent or non-covalentlinkages. Examples of adducts include, but are not limited to,indodicarbocyanine dye amidites, amino-substituted nucleotides, ethidiumbromide, ethidium homodimer, (1,3-propanediamino)propidium,(diethylenetriamino)propidium, thiazole orange,(N-N′-tetramethyl-1,3-propanediamino)propyl thiazole orange,(N-N′-tetramethyl-1,2-ethanediamino)propyl thiazole orange, thiazoleorange-thiazole orange homodimer (TOTO), thiazole orange-thiazole blueheterodimer (TOTAB), thiazole orange-ethidium heterodimer 1 (TOED1),thiazole orange-ethidium heterodimer 2 (TOED2) and fluorescein-ethidiumheterodimer (FED), psoralens, biotin, streptavidin, avidin, etc.

Where a first oligonucleotide is complementary to a region of a targetnucleic acid and a second oligonucleotide has complementary to the sameregion (or a portion of this region) a “region of overlap” exists alongthe target nucleic acid. The degree of overlap will vary depending uponthe nature of the complementarity (see, e.g., region “X” in FIGS. 29 and67 and the accompanying discussions).

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, recombinant Cleavasenucleases are expressed in bacterial host cells and the nucleases arepurified by the removal of host cell proteins; the percent of theserecombinant nucleases is thereby increased in the sample.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that comprises of segments of DNA joined together by means ofmolecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule that is expressed from a recombinantDNA molecule.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid (e.g., 4, 5, 6, . . . , n−1).

The term “nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin that may besingle or double stranded, and represent the sense or antisense strand.Similarly, “amino acid sequence” as used herein refers to peptide orprotein sequence.

The term “peptide nucleic acid” (“PNA”) as used herein refers to amolecule comprising bases or base analogs such as would be found innatural nucleic acid, but attached to a peptide backbone rather than thesugar-phosphate backbone typical of nucleic acids. The attachment of thebases to the peptide is such as to allow the bases to base pair withcomplementary bases of nucleic acid in a manner similar to that of anoligonucleotide. These small molecules, also designated anti geneagents, stop transcript elongation by binding to their complementarystrand of nucleic acid (Nielsen, et al. Anticancer Drug Des. 8:53 63[1993]).

As used herein, the terms “purified” or “substantially purified” referto molecules, either nucleic or amino acid sequences, that are removedfrom their natural environment, isolated or separated, and are at least60% free, preferably 75% free, and most preferably 90% free from othercomponents with which they are naturally associated. An “isolatedpolynucleotide” or “isolated oligonucleotide” is therefore asubstantially purified polynucleotide.

An isolated oligonucleotide (or polynucleotide) encoding a Pyrococcuswoesei (Pwo) FEN-1 endonuclease having a region capable of hybridizingto SEQ ID NO:116 is an oligonucleotide containing sequences encoding atleast the amino-terminal portion of Pwo FEN-1 endonuclease. An isolatedoligonucleotide (or polynucleotide) encoding a Pwo FEN-1 endonucleasehaving a region capable of hybridizing to SEQ ID NO:117 is anoligonucleotide containing sequences encoding at least thecarboxy-terminal portion of Pwo FEN-1 endonuclease. An isolatedoligonucleotide (or polynucleotide) encoding a Pwo FEN-1 endonucleasehaving a region capable of hybridizing to SEQ ID NOS:118 and 119 is anoligonucleotide containing sequences encoding at least portions of PwoFEN-1 endonuclease protein located internal to either the amino orcarboxy-termini of the Pwo FEN-1 endonuclease protein.

As used herein, the term “fusion protein” refers to a chimeric proteincontaining the protein of interest (e.g., Cleavase BN/thrombin nucleaseand portions or fragments thereof) joined to an exogenous proteinfragment (the fusion partner which consists of a non CleavaseBN/thrombin nuclease protein). The fusion partner may enhance solubilityof recombinant chimeric protein (e.g., the Cleavase BN/thrombinnuclease) as expressed in a host cell, may provide an affinity tag(e.g., a his-tag) to allow purification of the recombinant fusionprotein from the host cell or culture supernatant, or both. If desired,the fusion protein may be removed from the protein of interest (e.g.,Cleavase BN/thrombin nuclease or fragments thereof) by a variety ofenzymatic or chemical means known to the art.

As used herein, the terms “chimeric protein” and “chimerical protein”refer to a single protein molecule that comprises amino acid sequencesportions derived from two or more parent proteins. These parentmolecules may be from similar proteins from genetically distinctorigins, different proteins from a single organism, or differentproteins from different organisms. By way of example but not by way oflimitation, a chimeric structure-specific nuclease of the presentinvention may contain a mixture of amino acid sequences that have beenderived from FEN-1 genes from two or more of the organisms having suchgenes, combined to form a non-naturally occurring nuclease. The term“chimerical” as used herein is not intended to convey any particularproportion of contribution from the naturally occurring genes, nor limitthe manner in which the portions are combined. Any chimericstructure-specific nuclease constructs having cleavage activity asdetermined by the testing methods described herein are improved cleavageagents within the scope of the present invention.

The term “continuous strand of nucleic acid” as used herein is means astrand of nucleic acid that has a continuous, covalently linked,backbone structure, without nicks or other disruptions. The dispositionof the base portion of each nucleotide, whether base-paired,single-stranded or mismatched, is not an element in the definition of acontinuous strand. The backbone of the continuous strand is not limitedto the ribose-phosphate or deoxyribose-phosphate compositions that arefound in naturally occurring, unmodified nucleic acids. A nucleic acidof the present invention may comprise modifications in the structure ofthe backbone, including but not limited to phosphorothioate residues,phosphonate residues, 2′ substituted ribose residues (e.g., 2′-O-methylribose) and alternative sugar (e.g., arabinose) containing residues.

The term “continuous duplex” as used herein refers to a region of doublestranded nucleic acid in which there is no disruption in the progressionof basepairs within the duplex (i.e., the base pairs along the duplexare not distorted to accommodate a gap, bulge or mismatch with theconfines of the region of continuous duplex). As used herein the termrefers only to the arrangement of the basepairs within the duplex,without implication of continuity in the backbone portion of the nucleicacid strand. Duplex nucleic acids with uninterrupted basepairing, butwith nicks in one or both strands are within the definition of acontinuous duplex.

The term “duplex” refers to the state of nucleic acids in which the baseportions of the nucleotides on one strand are bound through hydrogenbonding the their complementary bases arrayed on a second strand. Thecondition of being in a duplex form reflects on the state of the basesof a nucleic acid. By virtue of base pairing, the strands of nucleicacid also generally assume the tertiary structure of a double helix,having a major and a minor groove. The assumption of the helical form isimplicit in the act of becoming duplexed.

The term “duplex dependent protein binding” refers to the binding ofproteins to nucleic acid that is dependent on the nucleic acid being ina duplex, or helical form.

The term “duplex dependent protein binding sites or regions” as usedherein refers to discrete regions or sequences within a nucleic acidthat are bound with particular affinity by specific duplex-dependentnucleic acid binding proteins. This is in contrast to the generalizedduplex-dependent binding of proteins that are not site-specific, such asthe histone proteins that bind chromatin with little reference tospecific sequences or sites.

The term “protein binding region” as used herein refers to a nucleicacid region identified by a sequence or structure as binding to aparticular protein or class of proteins. It is within the scope of thisdefinition to include those regions that contain sufficient geneticinformation to allow identifications of the region by comparison toknown sequences, but which might not have the requisite structure foractual binding (e.g., a single strand of a duplex-depending nucleic acidbinding protein site). As used herein “protein binding region” excludesrestriction endonuclease binding regions.

The term “complete double stranded protein binding region” as usedherein refers to the minimum region of continuous duplex required toallow binding or other activity of a duplex-dependent protein. Thisdefinition is intended to encompass the observation that some duplexdependent nucleic acid binding proteins can interact with full activitywith regions of duplex that may be shorter than a canonical proteinbinding region as observed in one or the other of the two singlestrands. In other words, one or more nucleotides in the region may beallowed to remain unpaired without suppressing binding. As used here in,the term “complete double stranded binding region” refers to the minimumsequence that will accommodate the binding function. Because some suchregions can tolerate non-duplex sequences in multiple places, althoughnot necessarily simultaneously, a single protein binding region mighthave several shorter sub-regions that, when duplexed, will be fullycompetent for protein binding.

The term “template” refers to a strand of nucleic acid on which acomplementary copy is built from nucleoside triphosphates through theactivity of a template-dependent nucleic acid polymerase. Within aduplex the template strand is, by convention, depicted and described asthe “bottom” strand. Similarly, the non-template strand is oftendepicted and described as the “top” strand.

The term “template-dependent RNA polymerase” refers to a nucleic acidpolymerase that creates new RNA strands through the copying of atemplate strand as described above and which does not synthesize RNA inthe absence of a template. This is in contrast to the activity of thetemplate-independent nucleic acid polymerases that synthesize or extendnucleic acids without reference to a template, such as terminaldeoxynucleotidyl transferase, or Poly A polymerase.

The term “ARRESTOR molecule” refers to an agent added to or included inan invasive cleavage reaction in order to stop one or more reactioncomponents from participating in a subsequent action or reaction. Thismay be done by sequestering or inactivating some reaction component(e.g., by binding or base-pairing a nucleic acid component, or bybinding to a protein component). The term “ARRESTOR oligonucleotide”refers to an oligonucleotide included in an invasive cleavage reactionin order to stop or arrest one or more aspects of any reaction (e.g.,the first reaction and/or any subsequent reactions or actions; it is notintended that the ARRESTOR oligonucleotide be limited to any particularreaction or reaction step). This may be done by sequestering somereaction component (e.g., base-pairing to another nucleic acid, orbinding to a protein component). However, it is not intended that theterm be so limited as to just situations in which a reaction componentis sequestered.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. inthe appropriate containers) and/or supporting materials (e.g., buffers,written instructions for performing the assay etc.) from one location toanother. For example, kits include one or more enclosures (e.g., boxes)containing the relevant reaction reagents and/or supporting materials.As used herein, the term “fragmented kit” refers to a delivery systemscomprising two or more separate containers that each contain asubportion of the total kit components. The containers may be deliveredto the intended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains oligonucleotides. The term “fragmented kit” isintended to encompass kits containing Analyte specific reagents (ASR's)regulated under section 520(e) of the Federal Food, Drug, and CosmeticAct, but are not limited thereto. Indeed, any delivery system comprisingtwo or more separate containers that each contain a subportion of thetotal kit components are included in the term “fragmented kit.” Incontrast, a “combined kit” refers to a delivery system containing all ofthe components of a reaction assay in a single container (e.g., in asingle box housing each of the desired components). The term “kit”includes both fragmented and combined kits.

As used herein, the term “functional domain” refers to a region, or apart of a region, of a protein (e.g., an enzyme) that provides one ormore functional characteristic of the protein. For example, a functionaldomain of an enzyme may provide, directly or indirectly, one or moreactivities of the enzyme including, but not limited to, substratebinding capability and catalytic activity. A functional domain may becharacterized through mutation of one or more amino acids within thefunctional domain, wherein mutation of the amino acid(s) alters theassociated functionality (as measured empirically in an assay) therebyindicating the presence of a functional domain.

As used herein, the term “heterologous functional domain” refers to aprotein functional domain that is not in its natural environment. Forexample, a heterologous functional domain includes a functional domainfrom one enzyme introduced into another enzyme. A heterologousfunctional domain also includes a functional domain native to an proteinthat has been altered in some way (e.g., mutated, added in multiplecopies, etc.). A heterologous functional domain may comprise a pluralityof contiguous amino acids or may include two or more distal amino acidsare amino acids fragments (e.g., two or more amino acids or fragmentswith intervening, non-heterologous, sequence). Heterologous functionaldomains are distinguished from endogenous functional domains in that theheterologous amino acid(s) are joined to amino acid sequences that arenot found naturally associated with the amino acid sequence in nature orare associated with a portion of a protein not found in nature.

As used herein, the term “altered functionality in a nucleic acidcleavage assay” refers to a characteristic of an enzyme that has beenaltered in some manner to differ from its natural state (e.g., to differfrom how it is found in nature). Alterations include, but are notlimited to, addition of a heterologous functional domain (e.g., throughmutation or through creation of chimerical proteins). In someembodiments, the altered characteristic of the enzyme may be one thatimproves the performance of an enzyme in a nucleic acid cleavage assay.Types of improvement include, but are not limited to, improved nucleaseactivity (e.g., improved rate of reaction), improved substrate binding(e.g., increased or decreased binding of certain nucleic acid species[e.g., RNA or DNA] that produces a desired outcome [e.g., greaterspecificity, improved substrate turnover, etc.]), and improvedbackground specificity (e.g., less undesired product is produced). Thepresent invention is not limited by the nucleic cleavage assay used totest improved functionality. However, in some preferred embodiments ofthe present invention, an invasive cleavage assay is used as the nucleicacid cleavage assay. In certain particularly preferred embodiments, aninvasive cleavage assay utilizing an RNA target is used as the nucleicacid cleavage assay.

As used herein, the terms “N-terminal” and “C-terminal” in reference topolypeptide sequences refer to regions of polypeptides includingportions of the N-terminal and C-terminal regions of the polypeptide,respectively. A sequence that includes a portion of the N-terminalregion of polypeptide includes amino acids predominantly from theN-terminal half of the polypeptide chain, but is not limited to suchsequences. For example, an N-terminal sequence may include an interiorportion of the polypeptide sequence including bases from both theN-terminal and C-terminal halves of the polypeptide. The same applies toC-terminal regions. N-terminal and C-terminal regions may, but need not,include the amino acid defining the ultimate N-terminal and C-terminalends of the polypeptide, respectively.

As used herein, the term “detection panel” refers to a substrate ordevice containing at least two unique candidate detection assaysconfigured for target detection.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a comparison of the nucleotide structure of the DNAP genesisolated from Thermus aquaticus (SEQ ID NO:1), Thermus flavus (SEQ IDNO:2) and Thermus thermophilus (SEQ ID NO:3); the consensus sequence(SEQ ID NO:7) is shown at the top of each row.

FIG. 2 is a comparison of the amino acid sequence of the DNAP isolatedfrom Thermus aquaticus (SEQ ID NO:4), Thermus flavus (SEQ ID NO:5), andThermus thermophilus (SEQ ID NO:6); the consensus sequence (SEQ ID NO:8)is shown at the top of each row.

FIGS. 3A-G are a set of diagrams of wild-type and synthesis-deficientDNAPTaq genes.

FIG. 4A depicts the wild-type Thermus flavus polymerase gene.

FIG. 4B depicts a synthesis-deficient Thermus flavus polymerase gene.

FIG. 5 depicts a structure which cannot be amplified using DNAPTaq; thisFigure shows SEQ ID NO:17 (primer) and SEQ ID NO:15 (hairpin).

FIG. 6 is a ethidium bromide-stained gel demonstrating attempts toamplify a bifurcated duplex using either DNAPTaq or DNAPStf (i.e., theStoffel fragment of DNAPTaq).

FIG. 7 is an autoradiogram of a gel analyzing the cleavage of abifurcated duplex by DNAPTaq and lack of cleavage by DNAPStf.

FIGS. 8A-B are a set of autoradiograms of gels analyzing cleavage orlack of cleavage upon addition of different reaction components andchange of incubation temperature during attempts to cleave a bifurcatedduplex with DNAPTaq.

FIGS. 9A-B are an autoradiogram displaying timed cleavage reactions,with and without primer.

FIGS. 10A-B are a set of autoradiograms of gels demonstrating attemptsto cleave a bifurcated duplex (with and without primer) with variousDNAPs.

FIG. 11A shows the substrate and oligonucleotides (19-12 [SEQ ID NO:18]and 30-12 [SEQ ID NO:19]) used to test the specific cleavage ofsubstrate DNAs targeted by pilot oligonucleotides.

FIG. 11B shows an autoradiogram of a gel showing the results of cleavagereactions using the substrates and oligonucleotides shown FIG. 12A.

FIG. 12A shows the substrate and oligonucleotide (30-0 [SEQ ID NO:20])used to test the specific cleavage of a substrate RNA targeted by apilot oligonucleotide.

FIG. 12B shows an autoradiogram of a gel showing the results of acleavage reaction using the substrate and oligonucleotide shown in FIG.13A.

FIG. 13 (SEQ ID NO:534) is a diagram of vector pTTQ18.

FIG. 14 (SEQ ID NO:535) is a diagram of vector pET-3c.

FIGS. 15A-E depicts a set of molecules which are suitable substrates forcleavage by the 5′ nuclease activity of DNAPs (SEQ ID NOS:15 and 17 aredepicted in FIG. 15E).

FIG. 16 is an autoradiogram of a gel showing the results of a cleavagereaction run with synthesis-deficient DNAPs.

FIG. 17 is an autoradiogram of a PEI chromatogram resolving the productsof an assay for synthetic activity in synthesis-deficient DNAPTaqclones.

FIG. 18A depicts the substrate molecule (SEQ ID NOS :15 and 22) used totest the ability of synthesis-deficient DNAPs to cleave short hairpinstructures.

FIG. 18B shows an autoradiogram of a gel resolving the products of acleavage reaction run using the substrate shown in FIG. 19A.

FIG. 19 provides the complete 206-mer duplex sequence (SEQ ID NO:27)employed as a substrate for the 5′ nucleases of the present invention

FIGS. 20A and B show the cleavage of linear nucleic acid substrates(based on the 206-mer of FIG. 21) by wild type DNAPs and 5′ nucleasesisolated from Thermus aquaticus and Thermus flavus.

FIG. 21A shows the “nibbling” phenomenon detected with the DNAPs of thepresent invention.

FIG. 21B shows that the “nibbling” of FIG. 25A is 5′ nucleolyticcleavage and not phosphatase cleavage.

FIG. 22 demonstrates that the “nibbling” phenomenon is duplex dependent.

FIG. 23 is a schematic showing how “nibbling” can be employed in adetection assay.

FIGS. 24A and B demonstrates that “nibbling” can be target directed.

FIG. 25 provides a schematic drawing of a target nucleic acid with anINVADER oligonucleotide and a probe oligonucleotide annealed to thetarget.

FIG. 26 provides a schematic showing the S-60 hairpin oligonucleotide(SEQ ID NO:29) with the annealed P-15 oligonucleotide (SEQ ID NO:30).

FIG. 27 is an autoradiogram of a gel showing the results of a cleavagereaction run using the S-60 hairpin in the presence or absence of theP-15 oligonucleotide.

FIG. 28 provides a schematic showing three different arrangements oftarget-specific oligonucleotides and their hybridization to a targetnucleic acid which also has a probe oligonucleotide annealed thereto(SEQ ID NOS:31-35).

FIG. 29 is the image generated by a fluorescence imager showing that thepresence of an INVADER oligonucleotide causes a shift in the site ofcleavage in a probe/target duplex.

FIG. 30 is the image generated by a fluorescence imager showing theproducts of INVADER oligonucleotide-directed cleavage assays run usingthe three target-specific oligonucleotides diagrammed in FIG. 28.

FIG. 31 is the image generated by a fluorescence imager showing theproducts of INVADER oligonucleotide-directed cleavage assays run in thepresence or absence of non-target nucleic acid molecules.

FIG. 32 is the image generated by a fluorescence imager showing theproducts of INVADER oligonucleotide-directed cleavage assays run in thepresence of decreasing amounts of target nucleic acid.

FIG. 33 is the image generated by a fluorescence imager showing theproducts of INVADER oligonucleotide-directed cleavage assays run in thepresence or absence of saliva extract using various thermostable 5′nucleases or DNA polymerases.

FIG. 34 is the image generated by a fluorescence imager showing theproducts of INVADER oligonucleotide-directed cleavage assays run usingvarious 5′ nucleases.

FIG. 35 is the image generated by a fluorescence imager showing theproducts of INVADER oligonucleotide-directed cleavage assays run usingtwo target nucleic acids which differ by a single basepair at twodifferent reaction temperatures.

FIG. 36A provides a schematic showing the effect of elevated temperatureupon the annealing and cleavage of a probe oligonucleotide along atarget nucleic acid wherein the probe contains a region ofnoncomplementarity with the target.

FIG. 36B provides a schematic showing the effect of adding an upstreamoligonucleotide upon the annealing and cleavage of a probeoligonucleotide along a target nucleic acid wherein the probe contains aregion of noncomplementarity with the target.

FIG. 37 provides a schematic showing an arrangement of a target-specificEVADER oligonucleotide (SEQ ID NO:39) and a target-specific probeoligonucleotide (SEQ ID NO:38) bearing a 5′ Cy3 label along a targetnucleic acid (SEQ ID NO:31).

FIG. 38 is the image generated by a fluorescence imager showing theproducts of INVADER oligonucleotide-directed cleavage assays run in thepresence of increasing concentrations of KCl.

FIG. 39 is the image generated by a fluorescence imager showing theproducts of INVADER oligonucleotide-directed cleavage assays run in thepresence of increasing concentrations of MnCl₂ or MgCl₂.

FIG. 40 is the image generated by a fluorescence imager showing theproducts of INVADER oligonucleotide-directed cleavage assays run in thepresence of increasing amounts of genomic DNA or tRNA.

FIG. 41 is the image generated by a fluorescence imager showing theproducts of INVADER oligonucleotide-directed cleavage assays run use aHCV RNA target.

FIG. 42 is the image generated by a fluorescence imager showing theproducts of INVADER oligonucleotide-directed cleavage assays run using aHCV RNA target and demonstrate the stability of RNA targets underINVADER oligonucleotide-directed cleavage assay conditions.

FIG. 43 is the image generated by a fluorescence imager showing thesensitivity of detection and the stability of RNA in INVADERoligonucleotide-directed cleavage assays run using a HCV RNA target.

FIG. 44 is the image generated by a fluorescence imager showing thermaldegradation of oligonucleotides containing or lacking a 3′ phosphategroup.

FIG. 45 depicts the structure of amino-modified oligonucleotides 70 and74.

FIG. 46 depicts the structure of amino-modified oligonucleotide 75

FIG. 47 depicts the structure of amino-modified oligonucleotide 76.

FIG. 48 is the image generated by a fluorescence imager scan of an IEFgel showing the migration of substrates 70, 70 dp, 74, 74 dp, 75, 75 dp,76 and 76 dp.

FIG. 49A provides a schematic showing an arrangement of atarget-specific INVADER oligonucleotide (SEQ ID NO:50) and atarget-specific probe oligonucleotide (SEQ ID NO:51) bearing a 5′ Cy3label along a target nucleic acid (SEQ ID NO:52).

FIG. 49B is the image generated by a fluorescence imager showing thedetection of specific cleavage products generated in an invasivecleavage assay using charge reversal (i.e., charge based separation ofcleavage products).

FIG. 50 is the image generated by a fluorescence imager which depictsthe sensitivity of detection of specific cleavage products generated inan invasive cleavage assay using charge reversal.

FIG. 51 depicts a first embodiment of a device for the charge-basedseparation of oligonucleotides.

FIG. 52 depicts a second embodiment of a device for the charge-basedseparation of oligonucleotides.

FIG. 53 shows an autoradiogram of a gel showing the results of cleavagereactions run in the presence or absence of a primer oligonucleotide; asequencing ladder is shown as a size marker.

FIGS. 54A-D depict four pairs of oligonucleotides; in each pair shown,the upper arrangement of a probe annealed to a target nucleic acid lacksan upstream oligonucleotide and the lower arrangement contains anupstream oligonucleotide (SEQ ID NOS:32 and 54-58 are shown in FIGS.54A-D).

FIG. 55 shows the chemical structure of several positively chargedheterodimeric DNA-binding dyes.

FIG. 56 is a schematic showing alternative methods for the tailing anddetection of specific cleavage products in the context of the INVADERoligonucleotide-directed cleavage assay.

FIG. 57 provides a schematic drawing of a target nucleic acid with anINVADER oligonucleotide, a miniprobe, and a stacker oligonucleotideannealed to the target.

FIG. 58 provides a space-filling model of the 3-dimensional structure ofthe T5 5′-exonuclease.

FIG. 59 provides an alignment of the amino acid sequences of severalFEN-1 nucleases including the Methanococcus jannaschii FEN-1 protein(MJAFEN1.PRO), the Pyrococcus furiosus FEN-1 protein (PFUFEN1.PRO), thehuman FEN-1 protein (HUMFEN1.PRO), the mouse FEN-1 protein(MUSFEN1.PRO), the Saccharomyces cerevisiae YKL510 protein (YST510.PRO),the Saccharomyces cerevisiae RAD2 protein (YSTRAD2.PRO), theShizosaccharomyces pombe RAD13 protein (SPORAD13.PRO), the human XPGprotein (HUMXPG.PRO), the mouse XPG protein (MUSXPG.PRO), the Xenopuslaevis XPG protein (XENXPG.PRO) and the C. elegans RAD2 protein(CELRAD2.PRO) (SEQ ID NOS:135-145, respectively); portions of the aminoacid sequence of some of these proteins were not shown in order tomaximize the alignment between proteins (specifically, amino acids 122to 765 of the YSTRAD2 sequence were deleted; amino acids 122 to 746 ofthe SPORAD13 sequence were deleted; amino acids 122 to 757 of the HUMXPGsequence were deleted; amino acids 122 to 770 of the MUSXPG sequencewere deleted; and amino acids 122 to 790 of the XENXPG sequence weredeleted). The numbers to the left of each line of sequence refers to theamino acid residue number; dashes represent gaps introduced to maximizealignment.

FIG. 60 is a schematic showing the S-33 (SEQ ID NO:84) and 11-8-0 (SEQID NO:85) oligonucleotides in a folded configuration; the cleavage siteis indicated by the arrowhead.

FIG. 61 shows a Coomassie stained SDS-PAGE gel showing the thrombindigestion of CLEAVASE BN/thrombin.

FIG. 62 is the image generated by a fluorescence imager showing theproducts produced by the cleavage of the S-60 hairpin using CLEAVASEBN/thrombin (before and after thrombin digestion).

FIG. 63 is the image generated by a fluorescence imager showing theproducts produced by the cleavage of circular M13 DNA using CLEAVASEBN/thrombin.

FIG. 64 is an SDS-PAGE gel showing the migration of purified CLEAVASE BNnuclease, Pfu FEN-1, Pwo FEN-1 and Mja FEN-1.

FIG. 65 is the image generated by a fluorescence imager showing theproducts produced by the cleavage of the S-33 and 11-8-0oligonucleotides by CLEAVASE BN and the Mja FEN-1 nucleases.

FIG. 66 (SEQ ID NO:86) is the image generated by a fluorescence imagershowing the products produced by the incubation of an oligonucleotideeither having or lacking a 3′-OH group with TdT.

FIG. 67 is the image generated by a fluorescence imager showing theproducts produced the incubation of cleavage products with TdT.

FIG. 68 is a photograph of a Universal GeneComb™ card showing thecapture and detection of cleavage products on a nitrocellulose support.

FIG. 69 is the image generated by a fluorescence imager showing theproducts produced using the CLEAVASE A/G and Pfu FEN-1 nucleases and afluorescein-labeled probe.

FIG. 70 is the image generated by a fluorescence imager showing theproducts produced using the CLEAVASE A/G and Pfu FEN-1 nucleases and aCy3-labeled probe;

FIG. 71 is the image generated by a fluorescence imager showing theproducts produced using the CLEAVASE A/G and Pfu FEN-1 nucleases and aTET-labeled probe.

FIGS. 72A and 72B are images generated by a fluorescence imager showingthe products produced using the CLEAVASE A/G and Pfu FEN-1 nucleases andprobes having or lacking a 5′ positive charge; the gel shown in FIG. 83Awas run in the standard direction and the gel shown in FIG. 84B was runin the reverse direction.

FIG. 73 shows the structure of 3-nitropyrrole and 5-nitroindole.

FIG. 74 shows the sequence of oligonucleotides 109, 61 and 67 (SEQ IDNOS:97, 50 and 51) annealed into a cleavage structure as well as thesequence of oligonucleotide 67 (SEQ ID NO:51) and a composite of SEQ IDNOS:98, 99, 101 and 102.

FIG. 75A-C show images generated by a fluorescence imager showing theproducts produced in an INVADER oligonucleotide-directed cleavage assayperformed at various temperatures using a miniprobe which is eithercompletely complementary to the target or contains a single mismatchwith the target.

FIG. 76 shows the sequence of oligonucleotides 166 (SEQ ID NO:103), 165(SEQ lID NO:104), 161 (SEQ ID NO:106), 162 (SEQ ID NO:105) and 164 (SEQID NO:107) as well as a cleavage structure showing the oligonucleotidesaligned with a consensus target seciuence (SEQ ID NO: 552).

FIG. 77 shows the image generated by a fluorescence imager showing theproducts produced in an INVADER oligonucleotide-directed cleavage assayperformed using ras gene sequences as the target.

FIGS. 78A-C show the sequence of the S-60 hairpin (SEQ ID NO:29) (A),and the P-15 oligonucleotide (SEQ ID NO:30) (shown annealed to the S-60hairpin in B) and the image generated by a fluorescence imager showingthe products produced by cleavage of the S-60 hairpin in the presence ofvarious INVADER oligonucleotides.

FIG. 79 shows the structure of various 3′ end substituents.

FIG. 80 is a composite graph showing the effect of probe concentration,temperature and a stacker oligonucleotide on the cleavage of miniprobes.

FIG. 81 shows the sequence of the IT-2 oligonucleotide (SEQ ID NO:115;shown in a folded configuration) as well as the sequence of the IT-1(SEQ ID NO:116) and IT-A (SEQ ID NO:117) oligonucleotides.

FIG. 82 shows the image generated by a fluorescence imager showing theproducts produced by cleavage of the oligonucleotides shown in FIG. 92by CLEAVASE A/G nuclease.

FIG. 83 shows the image generated by a fluorescence imager whichprovides a comparison of the rates of cleavage by the Pfu FEN-1 and MjaFEN-1 nucleases.

FIG. 84 shows the image generated by a fluorescence imager which depictsthe detection of RNA targets using a miniprobe and stackeroligonucleotides.

FIGS. 85A-C provide schematics showing particular embodiments of thepresent invention wherein a T7 promoter region and copy templateannealed with either no oligonucleotide (A), a complete promoteroligonucleotide (B) or a complete promoter oligonucleotide with a 3′tail (C); one strand of the T7 promoter region is indicated by thehatched line.

FIGS. 86A-D provide schematics showing particular embodiments of thepresent invention wherein a T7 promoter region and copy templateannealed with either a cut probe(A), a partial promoter oligonucleotide(B), an uncut oligonucleotide (C) or both an uncut probe and a partialpromoter oligonucleotide (D).

FIG. 87 provides a schematic illustrating one embodiment of the presentinvention wherein a template-dependent DNA polymerase is used to extenda cut probe to complete a T7 promoter region and thereby allowtranscription.

FIG. 88 provides a schematic illustrating that an uncut probe combinedwith a partial promoter oligonucleotide does not permit transcriptionwhile a cut probe combined with a partial promoter oligonucleotidegenerates a complete (but nicked) promoter which supports transcription.

FIG. 89 shows the image generated by a fluorescence imager which showsthat primer extension can be used to complete a partial promoter formedby a cut probe (lanes 1-5) and that annealing a cut probe generated inan invasive cleavage assay can complete a partial T7 promoter to permittranscription (lanes 6-9).

FIGS. 90A-C provide schematics showing particular embodiments of thepresent invention which illustrate that the use of a partial promoteroligonucleotide with a paired 5′ tail can be used to block transcriptionfrom a composite promoter formed by the annealing of an uncut probe.

FIG. 91 shows the image generated by a fluorescence imager which showsthat transcription from a “leaky” branched T7 composite promoter can beshut down by the use of a downstream partial promoter oligonucleotidehaving a paired 5′ tail.

FIG. 92 shows the image generated by a fluorescence imager which showsthat the location of the nick site in a nicked composite T7 promoter caneffect the efficiency of transcription.

FIG. 93 shows the image generated by a fluorescence imager which showsthat the presence of an unpaired 3′ tail on a full-length promoteroligonucleotide decreases but does not abolish transcription. Beneaththe image are schematics showing the nucleic acids tested in reactions1-4; these schematics show SEQ ID NOS:123-125.

FIG. 94 is a schematic which illustrates one embodiment of the presentinvention where a composite T7 promoter region is created by the bindingof the cut probe oligonucleotide downstream of the partial promoteroligo.

FIGS. 95A-D provide schematics showing particular embodiments of thepresent invention which show various ways in which a composite promotercan be formed wherein the nick is located in the template (or bottom)strand.

FIG. 96 is a schematic which illustrates one embodiment of the presentinvention where the cut probe from an initial invasive cleavage reactionis employed as the INVADER oligonucleotide in a second invasive cleavagereaction.

FIG. 97 is a schematic which illustrates one embodiment of the presentinvention where the cut probe from an initial invasive cleavage reactionis employed as an integrated INVADER-target complex in a second invasivecleavage reaction.

FIG. 98 shows the nucleotide sequence of the PR1 probe (SEQ ID NO:119),the IT3 INVADER-Target oligonculeotide (SEQ ID NO:118), the IT3-8,IT3-6, IT3-4, IT3-3 and IT3-0 oligonucleotides (SEQ ID NOS:147-151,respectively).

FIG. 99 depicts structures that may be employed to determine the ablityof an enzyme to cleave a probe in the presence and the absence of anupstream oligonucleotide. FIG. 99 displays the sequence ofoligonucleotide 89-15-1 (SEQ ID NO:152), oligonucleotide 81-69-5 (SEQ IDNO:156), oligonucleotide 81-69-4 (SEQ ID NO:155), oligonucleotide81-69-3 (SEQ ID NO:154), oligonucleotide 81-69-2 (SEQ ID NO:153) and aportion of M13mp18 (SEQ ID NO:163).

FIG. 100 shows the image generated by a fluorescence imager which showsthe dependence of Pfu FEN-1 on the presence of an overlapping upstreamoligonucleotide for specific cleavage of the probe.

FIG. 101 a shows the image generated by a fluorescence imager whichcompares the amount of product generated in a standard (i.e., anon-sequential invasive cleavage reaction) and a sequential invasivecleavage reaction.

FIG. 101 b is a graph comparing the amount of product generated in astandard or basic (i.e., a non-sequential invasive cleavage reaction)and a sequential invasive cleavage reaction (“invader sqrd”) (yaxis=fluorescence units; x axis=attomoles of target).

FIG. 102 shows the image generated by a fluorescence imager which showsthat the products of a completed sequential invasive cleavage reactioncannot cross contaminant a subsequent similar reaction.

FIG. 103 shows the sequence of the oligonucleotide employed in aninvasive cleavage reaction for the detection of HCMV viral DNA; FIG. 103shows the sequence of oligonucleotide 89-76 (SEQ ID NO:161),oligonucleotide 89-44 (SEQ ID NO:160) and nucleotides 3057-3110 of theHCMV genome (SEQ ID NO:162).

FIG. 104 shows the image generated by a fluorescence imager which showsthe sensitive detection of HCMV viral DNA in samples containing humangenomic DNA using an invasive cleavage reaction.

FIG. 105 is a schematic which illustrates one embodiment of the presentinvention, where the cut probe from an initial invasive cleavagereaction is employed as the INVADER oligonucleotide in a second invasivecleavage reaction, and where an ARRESTOR oligonucleotide preventsparticipation of remaining uncut first probe in the cleavage of thesecond probe.

FIG. 106 is a schematic which illustrates one embodiment of the presentinvention, where the cut probe from an initial invasive cleavagereaction is employed as an integrated INVADER-target complex in a secondinvasive cleavage reaction, and where an ARRESTOR oligonucleotideprevents participation of remaining uncut first probe in the cleavage ofthe second probe.

FIG. 107 shows three images generated by a fluorescence imager showingthat two different lengths of 2′ O-methyl, 3′ terminal amine-modifiedARRESTOR oligonucleotide both reduce non-specific background cleavage ofthe secondary probe when included in the second step of a reaction wherethe cut probe from an initial invasive cleavage reaction is employed asan integrated INVADER-target complex in a second invasive cleavagereaction.

FIG. 108A shows two images generated by a fluorescence imager showingthe effects on nonspecific and specific cleavage signal of increasingconcentrations of primary probe in the first step of a reaction wherethe cut probe from an initial invasive cleavage reaction is employed asthe INVADER oligonucleotide in a second invasive cleavage reaction.

FIG. 108B shows two images generated by a fluorescence imager showingthe effects on nonspecific and specific cleavage signal of increasingconcentrations of primary probe in the first step of a reaction, andinclusion of a 2′ O-methyl, 3′ terminal amine-modified ARRESTORoligonucleotide in the second step of a reaction where the cut probefrom an initial invasive cleavage reaction is employed as the INVADERoligonucleotide in a second invasive cleavage reaction.

FIG. 108C shows a graph generated using the spreadsheet Microsoft Excelsoftware, comparing the effects on nonspecific and specific cleavagesignal of increasing concentrations of primary probe in the first stepof a reaction, in the presence or absence of a 2′ O-methyl, 3′ terminalamine-modified ARRESTOR oligonucleotide in the second step of a reactionwhere the cut probe from an initial invasive cleavage reaction isemployed as the INVADER oligonucleotide in a second invasive cleavagereaction.

FIG. 109A shows two images generated by a fluorescence imager showingthe effects on nonspecific and specific cleavage signal of including anunmodified ARRESTOR oligonucleotide in the second step of a reactionwhere the cut probe from an initial invasive cleavage reaction isemployed as the INVADER oligonucleotide in a second invasive cleavagereaction.

FIG. 109B shows two images generated by a fluorescence imager showingthe effects on nonspecific and specific cleavage signal of including a3′ terminal amine modified ARRESTOR, a partially 2′ O-methylsubstituted, 3′ terminal amine modified ARRESTOR oligonucleotide, or anentirely 2′ O-methyl, 3′ terminal amine modified ARRESTORoligonucleotide in the second step of a reaction where the cut probefrom an initial invasive cleavage reaction is employed as the INVADERoligonucleotide in a second invasive cleavage reaction.

FIG. 110A shows two images generated by a fluorescence imager comparingthe effects on nonspecific and specific cleavage signal of including anARRESTOR oligonucleotides of different lengths in the second step of areaction where the cut probe from an initial invasive cleavage reactionis employed as the INVADER oligonucleotide in a second invasive cleavagereaction.

FIG. 110B shows two images generated by a fluorescence imager comparingthe effects on nonspecific and specific cleavage signal of including anARRESTOR oligonucleotides of different lengths in the second step of areaction where the cut probe from an initial invasive cleavage reactionis employed as the INVADER oligonucleotide in a second invasive cleavagereaction, and in which a longer variant of the secondary probe used inthe reactions in FIG. 110A is tested.

FIG. 110C shows a schematic diagram of a primary probe (SEQ ID NO: 536)aligned with several ARRESTOR oligonucleotides of different lengths. Theregion of the primary probe that is complementary to the HBV targetsequence is underlined. The ARRESTOR oligonucleotides (SEQ ID NOS:537-540) are aligned with the probe by complementarity.

FIG. 111 shows two images generated by a fluorescence imager comparingthe effects on nonspecific and specific cleavage signal of includingARRESTOR oligonucleotides of different lengths in the second step of areaction where the cut probe from an initial invasive cleavage reactionis employed as the INVADER oligonucleotide in a second invasive cleavagereaction, using secondary probes of two different lengths.

FIG. 112 A provides a schematic diagram that illustrates one embodimentof the present invention wherein the cut probe from an initial invasivecleavage reaction is employed as the INVADER oligonucleotide in a secondinvasive cleavage reaction using a FRET cassette. The region indicatedas “N” is the overlap required for cleavage in this embodiment. 112Bdiagrams how a mismatch between the probe and the target strand atposition “N” disrupts the overlap, thereby suppressing cleavage of theprobe.

FIG. 113A shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:195), probe oligonucleotide (SEQ ID NO:197) and FRET cassette (SEQID NO:201) for the detection of the Apo E 112 arg allele (SEQ IDNO:191). The flap released from the probe (SEQ ID NO:541) is shownannealed to the FRET cassette.

FIG. 113B shows a schematic diagram of an iNVADER oligonucleotide (SEQID NO:195), probe oligonucleotide (SEQ ID NO:198) and FRET cassette (SEQID NO:201) for the detection of the Apo E 112 cys allele (SEQ ID NO:192). The flap released from the probe (SEQ ID NO:542) is shown annealedto the FRET cassette.

FIG. 113C shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:196), probe oligonucleotide (SEQ ID NO:199) and FRET cassette (SEQID NO:201) for the detection of the Apo E 158 arg allele (SEQ ID NO:193)The flap released from the probe (SEQ ID NO:541) is shown annealed tothe FRET cassette.

FIG. 113D shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:196), probe oligonucleotide (SEQ ID NO:200) and FRET cassette (SEQID NO:201) for the detection of the Apo E 158 cys allele (SEQ IDNO:194). The flap released from the probe (SEQ ID NO:542) is shownannealed to the FRET cassette.

FIG. 114A provides a bar graph showing the detection of the arg and cysalleles at the Apo E 112 locus in 2 synthetic controls and 5 samples ofhuman genomic DNA.

FIG. 114B provides a bar graph showing the detection of the arg and cysalleles at the Apo E 158 locus in 2 synthetic controls and 5 samples ofhuman genomic DNA.

FIG. 115A shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:.202), probe oligonucleotide (SEQ ID NO:208) and FRET cassette(SEQ ID NO: 210) for the detection of the wild-type C282 allele of thehuman HFE gene (SEQ ID NO:204). The flap released from the probe (SEQ IDNO:543) is shown annealed to the FRET cassette.

FIG. 115B shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:202), probe oligonucleotide (SEQ ID NO:209) and FRET cassette (SEQID NO:210) for the detection of the C282Y mutant allele of the human HFEgene (SEQ ID NO:205). The flap released from the probe (SEQ ID NO:544)is shown annealed to the FRET cassette.

FIG. 115C shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:203), probe oligonucleotide (SEQ ID NO:211) and FRET cassette (SEQID NO:213) for the detection of the wild-type H63 allele of the humanEWE gene (SEQ ID NO:206) The flap released from the probe (SEQ IDNO:545) is shown annealed to the FRET cassette.

FIG. 115D shows a schematic diagram of an INVADER oligonucleotide (SEQID) NO:203), probe oligonucleotide (SEQ ID NO:212) and FRET cassette(SEQ ID NO:213) for the detection of the H63D mutant allele of the humanHFE gene (SEQ ID NO:207). The flap released from the probe (SEQ IDNO:546) is shown annealed to the FRET cassette.

FIG. 116 provides a bar graph showing the analysis of the C282Y (firstset of eight tests, left to right) and H63D (second set of eight tests,left to right) mutations in the human HFE gene, each tested in 2synthetic controls and 5 samples of human genomic DNA.

FIG. 117A shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:216), probe oligonucleotide (SEQ ID NO:217) and FRET cassette (SEQID NO:225) for the detection of the wild-type allele at position 677 ofthe human MTHFR gene (SEQ ID NO:214). The flap released from the probe(SEQ ID NO:541) is shown annealed to the FRET cassette.

FIG. 117B shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:216), probe oligonucleotide (SEQ ID NO:218) and FRET cassette (SEQID NO:225) for the detection of the mutant allele at position 677 of thehuman MTHFR gene (SEQ ID NO:215). The flap released from the probe (SEQID NO:542) is shown annealed to the FRET cassette.

FIG. 118 provides a bar graph showing the analysis of the C677T mutationin the human MTHFR gene in 3 synthetic control samples and 3 samples ofhuman genomic DNA.

FIG. 119A shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:222), probe oligonucleotide (SEQ ID NO:223) and FRET cassette (SEQID NO: 225) for the detection of the wild-type allele at position 20210of the human protbrombin gene (SEQ ID NO:220). The flap released fromthe probe (SEQ ID NO:541) is shown annealed to the FRET cassette.

FIG. 119B shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:222), probe oligonucleotide (SEQ ID NO:224) and FRET cassette (SEQID NO:225) for the detection of the mutant allele at position 20210 ofthe human prothrombin gene (SEQ ID NO:221). The flap released from theprobe (SEQ ID NO:542) is shown annealed to the FRET cassette.

FIG. 120 provides a bar graph showing the analysis of the A20210Gmutation in the human prothrombin gene in 2 synthetic control samplesand 3 samples of human genomic DNA.

FIG. 121A shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:228), probe oligonucleotide (SEQ ID NO:229) and FRET cassette (SEQID NO:230) for the detection of the R-2 mutant allele of the humanfactor V gene (SEQ ID NO:226). The flap released from the probe (SEQ IDNO:545) is shown annealed to the FRET cassette.

FIG. 121B shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:231), probe oligonucleotide (SEQ ID NO:232) and FRET cassette (SEQID NO: 230) for the detection of the human a-actin gene (SEQ ID NO:227).The flap released from the probe SEQ ID NO:547) is shown annealed to theFRET cassette.

FIG. 122 provides a bar graph showing the detection of the R-2 mutant(HR-2) of the human factor V gene, compared to the detection of theinternal control (IC), the α-actin gene, 3 synthetic control samples and2 samples of human genomic DNA.

FIG. 123A shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:235), probe oligonucleotide (SEQ ID NO:236) and FRET cassette (SEQID NO:225) for the detection of the wild-type allele at position -308 inthe promoter of the human TNF-αgene (SEQ NO:233). The flap released fromthe probe (SEQ ID) NO:548) is shown annealed to the FRET cassette.

FIG. 123B shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:235), probe oligonucleotide (SEQ ID NO:237) and FRET cassette (SEQID NO:225) for the detection of the mutant allele at position -308 inthe promoter of the human TNF-αgene (SEQ ID NO:234). The flap releasedfrom the probe (SEQ ID NO:549) is shown annealed to the FRET cassette.

FIG. 124 provides a bar graph showing the analysis of the -308 mutationin the promoter of the human TNF-αgene in 3 synthetic control samplesand 3 samples of human genomic DNA.

FIG. 125A shows a schematic diagram of an iNVADER oligonucleotide (SEQID NO:240), probe oligonucleotide (SEQ ID NO:241) and FRET cassette (SEQlID NO: 225) for the detection of the wild-type allele at codon position506 of the human factor V gene (SEQ ID NO:238). The flap released fromthe probe (SEQ ID NO:541) is shown annealed to the FRET cassette.

FIG. 125B shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:240), probe oligonucleotide (SEQ ID NO:242) and FRET cassette (SEQID NO:225) for the detection of the A506G mutant allele of the humanfactor V gene (SEQ ID NQ:239). The flap released from the probe (SEQ IDNO:542) is shown annealed to the FRET cassette.

FIG. 126 provides a bar graph showing the analysis of the A506G mutationin the human factor V gene in 3 synthetic control samples and 6 samplesof human genomic DNA.

FIG. 127A shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:243), probe oligonucleotide (SEQ ID NO:244) and FRET cassette (SEQID NQ:245) for the detection of the mecA gene associated withmethicillin resistance in S. aureus (SEQ ID NO:252). The flap releasedfrom the probe (SEQ ID NQ:550) is shown annealed to the FRET cassette.

FIG. 127B shows a schematic diagram of an INVADER oligonucleotide (SEQID NO:246), probe oligonucleotide (SEQ ID NO:247) and FRET cassette (SEQID NO:245) for the detection of the nuc gene, a species-specific genethat distinguishes S. aureus from S. haemolyticus (SEQ ID NO:253). Theflap released from the probe (SEQ ID NQ:551) is shown annealed to theFRET cassette.

FIG. 128 provides a bar graph showing the detection of the mecA gene,compared to the detection of the S. aureus-specific nuc gene in DNA frommethicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus(MRSA), S. haemolyticus, and amplified control targets for the mecA andnuc target sequences.

FIG. 129A shows the image generated by a fluorescence imager comparingthe products produced by cleavage of a mixture of the oligonucleotidesshown in FIG. 60 by either Pfu FEN-1 (1) or Mja FEN-1 (2).

FIG. 129B shows the image generated by a fluorescence imager comparingthe products produced by cleavage of the oligonucleotides shown in FIG.26 by either Pfu FEN-1 (1) or Mja FEN-1 (2).

FIG. 130 shows a schematic diagram of the portions of the Pfu FEN-1 andMja FEN-1 proteins combined to create chimeric nucleases.

FIG. 131A shows the image generated by a fluorescence imager comparingthe products produced by cleavage of a mixture of the oligonucleotidesshown in FIG. 60 by Pfu FEN-1 (1), Mja FEN-1 (2) or the chimericnucleases diagrammed in FIG. 130.

FIG. 131B shows the image generated by a fluorescence imager comparingthe products produced by cleavage of the oligonucleotides shown in FIG.26 by Pfu FEN-1 (1), Mja FEN-1 (2) or the chimeric nucleases diagrammedin FIG. 130.

FIG. 132 shows the image generated by a fluorescence imager comparingthe products produced by cleavage of folded cleavage structures by PfuFEN-1 (1), Mja FEN-1 (2) or the chimeric nucleases diagrammed in FIG.130.

FIG. 133A-J shows the results of various assays used to determine theactivity of Cleavase BN under various conditions.

FIG. 134A-B, D-F, and H-J show the results of various assays used todetermine the activity of TaqDN under various conditions.

FIG. 135A-B, D-F, H-J show the results of various assays used todetermine the activity of TthDN under various conditions.

FIG. 136A-B, D-F, and H-J show the results of various assays used todetermine the activity of Pfu FEN-1 under various conditions.

FIG. 137A-J show the results of various assays used to determine theactivity of Mja FEN-1 under various conditions.

FIG. 138A-B, D-F, and H-J show the results of various assays used todetermine the activity of Afu FEN-1 under various conditions.

FIGS. 139A-J show the results of various assays used to determine theactivity of Mth FEN-1 under various conditions.

FIG. 140 shows the two substrates. Panel A shows the structure andsequence of the hairpin substrate (25-65-1)(SEQ ID NO:293), while PanelB shows the structure and sequence of the INVADER (IT) substrate(25-184-5)(SEQ ID NO:294).

FIG. 141A shows the structure and sequence of oligonucleotides formingan invasive cleavage structure (203-91-01, SEQ ID NO:403, andtarget-INVADER oligonucleotide 203-91-04, SEQ ID NO:404).

FIG. 141B shows the structure and sequence of oligonucleotides formingan X-structure substrate (203-81-02, SEQ ID NO:405 and 594-09-01, SEQ IDNO:406).

FIG. 142 shows the activities of the -indicated FEN proteins on theinvasive cleavage structure diagrammed in FIG. 141A.

FIG. 143 shows the activities of the indicated FEN proteins on theX-structure diagrammed in FIG. 141B.

FIG. 144 shows a schematic diagram of an INVADER oligonucleotide (SEQ IDNO:407), probe oligonucleotide (SEQ ID NO:408) and FRET cassette (SEQ IDNO:409) for the detection of the polymerase gene of humancytomegalovirus.

FIG. 145 provides a bar graph showing the detection of different numbersof copies of human cytomegalovirus genomic DNA.

FIG. 146A-J, shown respectively as. SEQ ID NOS:378-379, 360-361,383-384, 336-337, 340-341, 344-345, 348-349, 369-370, 374-375, 388-389,352-353, 397-398, 393-394, 356-357, 364-365, and 401-402, shows nucleicacid and amino acid sequences for certain FEN-1 endonucleases of thepresent invention.

FIG. 147 shows a schematic diagram of one embodiment of ahigh-throughput enzyme screening system.

FIG. 148 shows graphs comparing cleavage rates observed using modifiedenzymes and INVADER oligonucleotides having different 3′ termini.

FIGS. 149A and 149B show overlap flap substrates designed for theactivity assays and MD simulations of Example 69. The substratescomprise upstream and downstream oligonucleotides annealed to a templateoligonucleotide. The 3′ end nucleotide of the upstream oligonucleotide,a thymine in this substrate, overlaps with the first base pair, G-C, ofthe downstream duplex thus creating the optimal substrate for thestructure-specific 5′ nucleases (Kaiser et al., 1999).

FIG. 149A (SEQ ID NOS:529-531) shows a schematic diagram of the overlapflap substrate used in FENi enzymatic assays. The substrate includes19-nt upstream and 17-nt downstream oligonucleotides annealed to a 40-nttemplate oligonucleotide. Base pairing is shown by vertical bars. The5′flap is single-stranded part of the downstream oligonucleotide.Nucleosides are numbered from the 5′end of the oligonucleotides andphosphates are assigned the same number as the adjacent 3′nucleoside.The specific FEN1cleavage site is shown by the arrow. Boxed nucleosidesand phosphates show positions of 2′-O-methyl and methylphosphonatemodifications, respectively, which affect the PfUFEN1cleavage rate. TETis tetracholorofluorecsein dye.

FIG. 149B (SEQ ID NOS:532-533) shows a schematic diagram of the overlapflap substrate used. in MD simulations. The structure was designed usingthe PfuFEN1 footprint identified in 150A. Nucleoside and phosphateresidues have the same notation as the corresponding residues in (A).

FIGS. 150A and B show schematic diagrams of phosphodiester andmethylphosphonate internucleoside linkages, respectively.

FIG. 151 shows the image generated by a fluorescence imager showing gelelectrophoresis analysis of PfuFEN1 cleavage products of the naturaloverlap flap substrate (N) and substrates with methylphosphonatesubstitutions at the template strand (A), upstream strand (B), ordownstream strand (C). The uncleaved downstream oligonucleotide (17-nt)and cleaved 5′ flap (5-nt) are indicated. Positions of methylphosphonatemodification are shown at the top of each panel. Reactions were carriedwith 0.1 nM PfuFEN1 and 50 nM substrate at 51° C. for 5 minutes. Forbrevity, modifications distant from the overlap site that have no effecton cleavage activity are not shown.

FIG. 152 shows Excel diagrams of the k_(cat)/K_(M) for PfuFEN1 onsubstrates with methylphosphonate substitutions at the template strand(A), upstream strand (B), or downstream strand (C) normalized fork_(cat)/K_(M) of the natural substrate. Results are shown only formodifications closest to the overlap site. Coefficient of variation forthe relative k_(cat)/K_(M) values determined from four independentexperiments was 25%. The positions of substitution are indicated beloweach bar, with the position numbering as shown in FIG. 149.

FIG. 153 shows Excel diagrams of the k_(cat)/K_(M) values for PfuFEN1 onsubstrates with 2′-O-methyl substitutions at the template strand (A),upstream strand (B), or downstream strand (C) normalized for kcat/KM ofthe natural substrate. Results are shown only for modifications closestto the overlap site. The coefficient of variation for the relativek_(cat)/K_(M) values determined from four independent experiments was25%. The positions of substitution are indicated below each bar, withthe position numbering as shown in FIG. 149.

FIG. 154 shows a computer-generated model of 1-ns structures ofPfuFEN1/DNA complexes in orientations I (A) and II (B). For bothorientations, the cleaved phosphodiester bond Pd6 of the DNA wasrestrained at distance of 2.5 to 4.5 Å from the M-1 magnesium ion. Thetemplate, upstream and downstream oligonucleotides are shown in green,magenta, and blue, respectively. In orientation I, the DNA is located inthe DNA-binding groove with the downstream duplex (green-blue) facingthe HhH motif (red) of the enzyme. In orientation II, the DNA wasrotated 180° relative to the position of the downstream and upstreamduplexes in the DNA-binding groove.

FIG. 155 shows Excel diagrams of the relative k_(cat)/K_(M) values forR64A (A), R94A (B) and PfuM3 (C) mutants on the substrates withmethylphosphonate substitutions. The k_(cat)/K_(M) values werenormalized for that of the natural substrate. Results are shown only forregions where changes in k_(cat)/K_(M) profile for each of the mutantsoccurred. Reactions were performed with 50 nM substrate using 0.5 nMenzyme at 51 oC for 10 minutes. The coefficient of variation of therelative k_(cat)/K_(M) values determined from four independentexperiments with the natural substrate was 25%. The positions ofsubstitution are indicated below each bar, with the position numberingas shown in FIG. 149.

FIG. 156 shows computer-generated 5 ns MD model of the PfuFEN1/DNAcomplex with residue-specific restraints. A, a view showing the overlapflap substrate bound in the DNA-binding groove of PfuFEN1. The HhHmotif, bA/bB hairpin, helical arch and magnesium ions are shown in red,orange, yellow and pink, respectively. Arginines 94 and 64 are displayedwith a space filled model. The template, upstream and downstreamoligonucleotides are shown in green, magenta, and blue, respectively. B,a 90 degree rotated view of the model shown in A. C, a cartoondescription of the substrate structure in the model (adopted from theview A). The 3′ end overlapping base of the upstream oligonucleotide isshown by arrows. The phosphodiester linkages P_(d)5, P_(d)6, P_(t)12,P_(t)13, P_(t)17, P_(t)18, P_(t)21, and P_(u)19 are shown withasterisks.

FIG. 157 shows a schematic representation of interactions proposed inthe PfuFEN1/DNA complex. Hydrogen bonds and ion pairs are shown by solidand dashed lines, respectively. Backbone nitrogens of the G244, G246,K248, and K249 amino acids in the HhH motif form hydrogen bonds withP_(t)12 and P_(t)13 in the template oligonucleotide. Amino acid R94forms ion pairs with Pd5 and Pd6 in the downstream oligonucleotide andamino acid R64 contacts P_(t)21 in the template oligonucleotide. ThebA/bB loop interacts with the upstream duplex between residues P_(t)21and P_(t)22.

FIG. 158 shows Excel diagrams of the comparing the activities of PfuFEN1with the R94 mutants on the natural DNA and P_(d)6 methylphosphonatesubstituted substrates.

DESCRIPTION OF THE INVENTION Introduction

The present invention relates to methods and compositions for treatingnucleic acid, and in particular, methods and compositions for detectionand characterization of nucleic acid sequences and sequence changes.

In preferred embodiments, the present invention relates to means forcleaving a nucleic acid cleavage structure in a site-specific manner.While the present invention provides a variety of cleavage agents, insome embodiments, the present invention relates to a cleaving enzymehaving 5′ nuclease activity without interfering nucleic acid syntheticability. In other embodiments, the present invention provides novelpolymerases (e.g., thermostable polymerases) possessing alteredpolymerase and/or nucleases activities.

For example, in some embodiments, the present invention provides 5′nucleases derived from thermostable DNA polymerases that exhibit alteredDNA synthetic activity from that of native thermostable DNA polymerases.The 5′ nuclease activity of the polymerase is retained while thesynthetic activity is reduced or absent. Such 5′ nucleases are capableof catalyzing the structure-specific cleavage of nucleic acids in theabsence of interfering synthetic activity. The lack of syntheticactivity during a cleavage reaction results in nucleic acid cleavageproducts of uniform size.

The novel properties of the nucleases of the invention form the basis ofa method of detecting specific nucleic acid sequences. This methodrelies upon the amplification of the detection molecule rather than uponthe amplification of the target sequence itself as do existing methodsof detecting specific target sequences.

DNA polymerases (DNAPs), such as those isolated from E. coli or fromthermophilic bacteria of the genus Thermus as well as other organisms,are enzymes that synthesize new DNA strands. Several of the known DNAPscontain associated nuclease activities in addition to the syntheticactivity of the enzyme.

Some DNAPs are known to remove nucleotides from the 5′ and 3′ ends ofDNA chains (Kornberg, DNA Replication, W. H. Freeman and Co., SanFrancisco, pp. 127-139 [1980]). These nuclease activities are usuallyreferred to as 5′ exonuclease and 3′ exonuclease activities,respectively. For example, the 5′ exonuclease activity located in theN-terminal domain of several DNAPs participates in the removal of RNAprimers during lagging strand synthesis during DNA replication and theremoval of damaged nucleotides during repair. Some DNAPs, such as the E.coli DNA polymerase (DNAPEc1), also have a 3′ exonuclease activityresponsible for proof-reading during DNA synthesis (Kornberg, supra).

A DNAP isolated from Thermus aquaticus, termed Taq DNA polymerase(DNAPTaq), has a 5′ exonuclease activity, but lacks a functional 3′exonucleolytic domain (Tindall and Kunkell, Biochem., 27:6008 [1988]).Derivatives of DNAPEc1 and DNAPTaq, respectively called the Klenow andStoffel fragments, lack 5′ exonuclease domains as a result of enzymaticor genetic manipulations (Brutlag et al., Biochem. Biophys. Res.Commun., 37:982 [1969]; Erlich et al., Science 252:1643 [1991]; Setlowand Kornberg, J. Biol. Chem., 247:232 [1972]).

The 5′ exonuclease activity of DNAPTaq was reported to requireconcurrent synthesis (Gelfand, PCR Technology—Principles andApplications for DNA Amplification, H. A. Erlich, [Ed.], Stockton Press,New York, p. 19 [1989]). Although mononucleotides predominate among thedigestion products of the 5′ exonucleases of DNAPTaq and DNAPEc1, shortoligonucleotides (≦12 nucleotides) can also be observed implying thatthese so-called 5′ exonucleases can function endonucleolytically(Setlow, supra; Holland et al., Proc. Natl. Acad. Sci. USA 88:7276[1991]).

In WO 92/06200, Gelfand et al. show that the preferred substrate of the5′ exonuclease activity of the thermostable DNA polymerases is displacedsingle-stranded DNA. Hydrolysis of the phosphodiester bond occursbetween the displaced single-stranded DNA and the double-helical DNAwith the preferred exonuclease cleavage site being a phosphodiester bondin the double helical region. Thus, the 5′ exonuclease activity usuallyassociated with DNAPs is a structure-dependent single-strandedendonuclease and is more properly referred to as a 5′ nuclease.Exonucleases are enzymes that cleave nucleotide molecules from the endsof the nucleic acid molecule. Endonucleases, on the other hand, areenzymes that cleave the nucleic acid molecule at internal rather thanterminal sites. The nuclease activity associated with some thermostableDNA polymerases cleaves endonucleolytically but this cleavage requirescontact with the 5′ end of the molecule being cleaved. Therefore, thesenucleases are referred to as 5′ nucleases.

When a 5′ nuclease activity is associated with a eubacterial Type A DNApolymerase, it is found in the one third N-terminal region of theprotein as an independent functional domain. The C-terminal two-thirdsof the molecule constitute the polymerization domain that is responsiblefor the synthesis of DNA. Some Type A DNA polymerases also have a 3′exonuclease activity associated with the two-third C-terminal region ofthe molecule.

The 5′ exonuclease activity and the polymerization activity of DNAPs canbe separated by proteolytic cleavage or genetic manipulation of thepolymerase molecule. The Klenow or large proteolytic cleavage fragmentof DNAPEc1 contains the polymerase and 3′ exonuclease activity but lacksthe 5′ nuclease activity. The Stoffel fragment of DNAPTaq (DNAPStf)lacks the 5′ nuclease activity due to a genetic manipulation thatdeleted the N-terminal 289 amino acids of the polymerase molecule(Erlich et al., Science 252:1643 [1991]). WO 92/06200 describes athermostable DNAP with an altered level of 5′ to 3′ exonuclease. U.S.Pat. No. 5,108,892 describes a Thermus aquaticus DNAP without a 5′ to 3′exonuclease. Thermostable DNA polymerases with lessened amounts ofsynthetic activity are available (Third Wave Technologies, Madison,Wis.) and are described in U.S. Pat. Nos. 5,541,311, 5,614,402,5,795,763, 5,691,142, and 5,837,450, herein incorporated by reference intheir entireties. The present invention provides 5′ nucleases derivedfrom thermostable Type A DNA polymerases that retain 5′ nucleaseactivity but have reduced or absent synthetic activity. The ability touncouple the synthetic activity of the enzyme from the 5′ nucleaseactivity proves that the 5′ nuclease activity does not requireconcurrent DNA synthesis as was previously reported (Gelfand, PCRTechnology, supra).

In addition to the 5′-exonuclease domains of the DNA polymerase Iproteins of Eubacteria, described above, 5′ nucleases have been foundassociated with bacteriophage, eukaryotes and archaebacteria. Overall,all of the enzymes in this family display very similar substratespecificities, despite their limited level of sequence similarity.Consequently, enzymes suitable for use in the methods of the presentinvention may be isolated or derived from a wide array of sources.

A mammalian enzyme with functional similarity to the 5′-exonucleasedomain of E. coli Pol I was isolated nearly 30 years ago (Lindahl, etal., Proc Natl Acad Sci U S A 62(2): 597-603 [1969]). Later, additionalmembers of this group of enzymes called flap endonucleases (FEN1) fromEukarya and Archaea were shown to possess a nearly identical structurespecific activity (Harrington and Lieber. Embo J 13(5), 1235-46 [1994];Murante et al., J Biol Chem 269(2), 1191-6 [1994]; Robins, et al., JBiol Chem 269(46), 28535-8 [1994]; Hosfield, et al., J Biol Chem273(42), 27154-61 [1998]), despite limited sequence similarity. Thesubstrate specificities of the FEN1 enzymes, and the eubacterial andrelated bacteriophage enzymes have been examined and found to be similarfor all enzymes (Lyamichev, et al., Science 260(5109), 778-83 [1993],Harrington and Lieber, supra, Murante, et al., supra, Hosfield, et al,supra, Rao, et al., J Bacteriol 180(20), 5406-12 [1998], Bhagwat, etal,. J. Biol Chem 272(45), 28523-30 [1997], Garforth and Sayers, NucleicAcids Res 25(19), 3801-7 [1997]).

Using preformed substrates, many of the studies cited above determinedthat these nucleases leave a gap upon cleavage, leading the authors tospeculate that DNA polymerase must then act to fill in that gap togenerate a ligatable nick. A number of other 5′ nucleases have beenshown to leave a gap or overlap after cleavage of the same or similarflap substrates. It has since been determined that that all thestructure-specific 5′-exonucleases leave a nick after cleavage if thesubstrate has an overlap between the upstream and downstream duplexes(Kaiser et al., J. Biol Chem. 274(30):21387-21394 [1999]). Whileduplexes having several bases of overlapping sequence can assume severaldifferent conformations through branch migration, it was determined thatcleavage occurs in the conformation where the last nucleotide at the 3′end of the upstream strand is unpaired, with the cleavage rate beingessentially the same whether the end of the upstream primer is A, C, G,or T. It was determined to be positional overlap between the 3′ end ofthe upstream primer and downstream duplex, rather then sequence overlap,that is required for optimal cleavage. In addition to allowing theseenzymes to leave a nick after cleavage, the single base of overlapcauses the enzymes to cleave several orders of magnitude faster thanwhen a substrate lacks overlap (Kaiser et al., supra).

Any of the 5′ nucleases described above may find application in one ormore embodiments of the methods described herein. FEN1 nucleases ofparticular utility in the methods of present invention include but arenot limited to those of Methanococcus jannaschii and Methanobacteriumthermoautotrophicum; particularly preferred FEN1 enzymes are fromArchaeoglobus fulgidus, Pyrococcus furiosus, Archaeoglobus veneficus,Sulfolobus solfataricus, Pyrobaculum aerophilum, Thermococcus litoralis,Archaeaglobus veneficus, Archaeaglobus profundus, Acidianus brierlyi,Acidianus ambivalens, Desulfurococcus amylolyticus, Desulfurococcusmobilis, Pyrodictium brockii, Thermococcus gorgonarius, Thermococcuszilligii, Methanopyrus kandleri, Methanococcus igneus, Pyrococcushorikoshii, and Aeropyrum pernix.

Structure-specific 5′ nucleases have been isolated from differentorganisms including bacteriophages (Hollingsworth & Nossal, 1991; Sayers& Eckstein, 1990), eubacteria (Deutscher & Kornberg, 1969; Klenow &Overgaard-Hansen, 1970; Lyamichev et al., 1993), archaea (Hosfield etal., 1998a; Hwang et al., 1998; Kaiser et al., 1999), yeast (Habraken etal., 1993; Harrington & Lieber, 1994b) and mammals (Harrington & Lieber,1994a; Lindahl et al., 1969; Murante et al., 1994; O'Donovan et al.,1994b). The ubiquitous presence of these enzymes is explained by theiressential role in Okazaki fragment processing (Goulian et al., 1990;Lundquist & Olivera, 1982; Turchi & Bambara, 1993) and repair of DNAdamage caused by alkylating agents or UV radiation (Habraken et al.,1993; Murray et al., 1994; O'Donovan et al., 1994a). In Okazaki fragmentprocessing, displacement synthesis by DNA polymerases generates branchedDNA structures in which the upstream and downstream strands overlap andcompete for the same sequence of the template strand. The branchedstructure can exist in multiple conformations depending on the positionof the branch point between the upstream and downstream strands on theshared template sequence (Lundquist & Olivera, 1982; Reynaldo et al.,2000). The structure-specific 5′ nucleases known as flap endonucleases(FEN1) (Harrington & Lieber, 1994a) specifically recognize aconformation called the overlap flap or 3′ one-nucleotide double-flapstructure in which the upstream strand, excluding the 3′ end nucleotide,is annealed to the template strand, displacing the 5′ portion of thedownstream strand (Kaiser et al., 1999; Kao et al., 2002; Lyamichev etal., 1999). FEN1 cleaves the downstream strand of the overlap flapstructure precisely after the first base-paired nucleotide, creating aligatable nick (Kaiser et al., 1999; Kao et al., 2002).

The conclusion that the 3′ end nucleotide of the upstream strand is notbase-paired with the template has been put forth from the observationthat any of the four natural bases at this position can supportefficient cleavage (Lyamichev et al., 1999). It was suggested that the3′ end nucleotide of the upstream strand interacts with the enzyme toposition the substrate in an optimal orientation for cleavage. Thedemonstration that sugar modifications of the 3′ end nucleotide inhibitactivity of FEN1 enzymes has further supported this hypothesis (Kaiseret al., 1999; Kao et al., 2002). The importance of the overlapping 3′end nucleotide of the upstream strand was not originally recognized, andmany laboratories characterized FEN1 enzymes using a flap structurewhich included adjacent upstream and downstream strands annealed to atemplate, but lacking a gap or overlap (Harrington & Lieber, 1994b).FEN1 enzymes cleave such a flap substrate inefficiently producing amajority of products that are not ligatable. The existence of theseproducts can be explained by the formation of alternative structureswith bulged nucleotides stabilized by the 5′ nuclease in an effort toforce the overlap flap structure (Kaiser et al., 1999; Kao et al., 2002;Lyamichev et al., 1999).

X-ray crystal structures have been determined for six structure-specific5′ nucleases: the archaeal FEN1 enzymes from Pyrococcus furiosus(PfuFEN1) (Hosfield et al., 1998b), Methanococcus jannaschii (MjaFEN1)(Hwang et al., 1998) and Pyrococcus horikoshii (PhoFEN1) (Matsui et al.,2002); the 5′ nuclease domain of eubacterial DNA polymerase from Thermusaquaticus (TaqExo) (Kim et al., 1995); the 5′-3′ exonuclease frombacteriophage T5 (Ceska et al., 1996), and the RNase H enzyme frombacteriophage T4 (Mueser et al., 1996). These structures reveal a commona/b topology and similar structural motifs despite a low amino acidsequence identity and similarity between the archaeal, eubacterial andbacteriophage FEN1 groups (see, for example, (Hosfield et al., 1998b)).All of these enzymes have been shown to bind two divalent metal ionswhich form a complex network of interactions with highly conservedacidic amino acids lying at the bottom of a positively charged cleft.One metal ion is presumably involved in catalysis and the other in theDNA binding (Shen et al., 1996). In the PfuFEN1 structure, the magnesiumion M-1, involved in catalysis, is located in close proximity to thecluster of amino acids Asp27, Asp80, Glu152, and Glu154; and themagnesium ion M-2 involved in substrate binding interacts with thecluster of amino acids Asp 173, Asp 175, and Asp236 approximately 5 Åfrom M-1.

The helical arch is a common structural motif shared by the FEN1 enzymesand was originally identified in the T5 5′-3′ exonuclease structure(Ceska et al., 1996). The arch is located close to the enzyme's activesite and forms a flexible loop that can accommodate single-stranded butnot double-stranded DNA. The motif provides structural support for thehypothesis that the 5′ flap of DNA substrate threads through a hole totranslocate DNA to the enzyme's active site. The threading mechanism wasoriginally proposed to explain biochemical data that blocking the free5′ end of the flap with a bulky modification or rendering itdouble-stranded using a complementary oligonucleotide suppresses thecleavage efficiency and can even trap FEN1 on the 5′ flap (Lyamichev etal., 1993; Murante et al., 1995). While most studies agree on thethreading mechanism, Bambara and his group have shown that a variety ofbulky flap modifications can be tolerated by human FEN1 endonuclease(Bornarth et al., 1999).

Another common fold shared by the FEN1 enzymes is thehelix-hairpin-helix (HhH) motif found in many enzyme families (Dohertyet al., 1996; Shao & Grishin, 2000). This type of fold is involved innon-sequence-specific binding of duplex DNA via interactions with thesugar-phosphate backbone of one of the strands (Pelletier et al., 1996;Thayer et al., 1995). Together with the helical arch and network ofamino acids interacting with the M-1 and M-2 ions, the HhH motif definesa positively charged active-site DNA-binding groove in FEN1. In PfuFEN1,the DNA-binding groove is 32 Å wide and 44 Å long, suggesting that itcan accommodate a 12 base-pair double-stranded DNA (Hosfield et al.,1998b). Biochemical analysis of point mutations at the DNA-bindinggroove of the FEN1 enzymes revealed conserved amino acids on the surfaceof the groove involved in catalysis and substrate binding (Bhagwat etal., 1997; Garforth et al., 1999; Matsui et al., 2002; Qiu et al., 2002;Shen et al., 1996; Shen et al., 1997; Xu et al., 1997).

Structural and functional similarity between the 5′ nucleases suggests acommon mechanism for substrate binding and catalysis for all enzymes inthis family. In the absence of co-crystal or NMR structures for a 5′nuclease/DNA complex, several models of the complex have been proposedto elucidate the mechanism of substrate binding (Ceska et al., 1996;Dervan et al., 2002; Hosfield et al., 1998b; Hwang et al., 1998). Thesemodels suggest that the substrate binds at the active-site DNA-bindinggroove with the cleavable phosphodiester linkage close to the metal ioninvolved in catalysis and with the 5′ flap threading through the helicalarch.

Methylphosphonate and 2′-O-methyl substitutions have proven to bepowerful methods for identifying contacts between nucleic acids andproteins (Botfield & Weiss, 1994; Dertinger & Uhlenbeck, 2001; Hou etal., 2001; Noble et al., 1984; Pritchard et al., 1994; Smith &McLaughlin, 1997). Methylphosphonate substitutions are almost isostericwith phosphodiester linkages but unlike phosphodiester linkages areneutral and therefore can be used to identify ionic interactions inprotein/substrate complexes without introducing steric clashes with theproteins (Dertinger & Uhlenbeck, 2001). Methylphosphonate linkages havebeen shown to induce local bending in the double helical DNA axis by themechanism of asymmetric phosphate charge neutralization. However, thebending angle estimated as 3.5 o per methylphosphonate substitution(Tomky et al., 1998) is comparable to the intrinsic sequence-specificDNA bending (Goodsell et al., 1993) and thermal flexibility of duplexDNA of ˜7 o per base pair estimated from its persistence length (Cantor& Schimmel, 1980). Substitution of a methyl group in place of anon-bridging oxygen in the phosphodiester linkage at a point ofelectrostatic contact with a protein usually decreases the affinity ofsubstrate binding (Dertinger & Uhlenbeck, 2001). This property ofmethylphosphonate modifications makes unnecessary, in most cases, theseparation of Rp and Sp stereoisomers of chemically introducedmethylphosphonate linkages and justifies the use of their racemicmixtures. 2′-O-methyl substitutions replace the 2′ proton in thedeoxyribose ring with a bulky O-methyl group with two major outcomes forduplex DNA structure. First, 2′-O-methyl groups change theconformational preference of ribose from C2′-endo to C3′-endo sugarpuckering, forcing a local transition from B-form to A-form DNA. Second,they introduce steric clashes at sites of contacts with the proteins(Hou et al., 2001).

In work conducted during the development of the present invention, asingle methylphosphonate substitution was introduced into eachphosphodiester linkage of the overlap flap DNA substrate to mapphosphates interacting with PfuFEN1. Similarly, 2′-O-methylsubstitutions were introduced to identify steric contacts in thePfuFEN1/DNA complex. Using the three-dimensional structure of PfuFEN1(Hosfield et al., 1998b) and a modeled structure of the overlap flapsubstrate, energy minimization and molecular dynamics (MD) simulationswere performed to test two alternative structures of the PfuFEN1/DNAcomplex. The model consistent with the methylphosphonate data was usedto identify candidate amino acids contacting phosphates in thesubstrate. To confirm the predicted interactions, PfuFEN1 variantsmutated at these amino acids were tested on the methylphosphonatesubstrates. The confirmed interactions were used as restraints in MDsimulations to develop a detailed model of the PfuFEN1/DNA complex.

The detailed description of the invention is presented in the followingsections:

-   I. Detection of Specific Nucleic Acid Sequences Using 5′ Nucleases    in an INVADER Directed Cleavage Assay;-   II. Effect of ARRESTOR Oligonucleotides on Signal and Background in    Sequential Invasive Cleavage Reactions.-   III. Signal Enhancement By Incorporating The Products Of An Invasive    Cleavage Reaction Into A Subsequent Invasive Cleavage Reaction;-   IV. Fractionation Of Specific Nucleic Acids By Selective Charge    Reversal;-   V. Signal Enhancement By Tailing Of Reaction Products In The INVADER    oligonucleotide-directed Cleavage Assay;-   VI. Signal Enhancement By Completion Of An Activated Protein Binding    Site;-   VII. Generation of 5′ Nucleases Derived From Thermostable DNA    Polymerases;-   VIII. Improved Enzymes For Use In INVADER oligonucleotide-directed    Cleavage Reactions;-   IX. FEN Endonuclease-Substrate Complexes-   X. The INVADER assay for direct detection and measurement of    specific analytes.-   XI. Kits    I. Detection of Specific Nucleic Acid Sequences Using 5′ Nucleases    in an INVADER Directed Cleavage Assay

1. INVADER Assay Reaction Design

The present invention provides means for forming a nucleic acid cleavagestructure that is dependent upon the presence of a target nucleic acidand cleaving the nucleic acid cleavage structure so as to releasedistinctive cleavage products. 5′ nuclease activity, for example, isused to cleave the target-dependent cleavage structure and the resultingcleavage products are indicative of the presence of specific targetnucleic acid sequences in the sample. When two strands of nucleic acid,or oligonucleotides, both hybridize to a target nucleic acid strand suchthat they form an overlapping invasive cleavage structure, as describedbelow, invasive cleavage can occur. Through the interaction of acleavage agent (e.g., a 5′ nuclease) and the upstream oligonucleotide,the cleavage agent can be made to cleave the downstream oligonucleotideat an internal site in such a way that a distinctive fragment isproduced. Such embodiments have been termed the INVADER assay (ThirdWave Technologies) and are described in U.S. Pat. Nos. 5,846,717;6,001,567; 5,985,557; 5,994,069; 6,090,543; 6,348,314; 6,458,535; U.S.patent application Nos. 20030186238 (Ser. No. 10/084839); 20030104378A1(Ser. No. 09/864636); Lyamichev et al., Nat. Biotech., 17:292 (1999),Hall et al., PNAS, USA, 97:8272 (2000), WO97/27214 and WO98/42873, eachof which is herein incorporated by reference in its entirety for allpurposes.

The present invention further provides assays in which the targetnucleic acid is reused or recycled during multiple rounds ofhybridization with oligonucleotide probes and cleavage of the probeswithout the need to use temperature cycling (i.e., for periodicdenaturation of target nucleic acid strands) or nucleic acid synthesis(i.e., for the polymerization-based displacement of target or probenucleic acid strands). When a cleavage reaction is run under conditionsin which the probes are continuously replaced on the target strand (e.g.through probe-probe displacement or through an equilibrium betweenprobe/target association and disassociation, or through a combinationcomprising these mechanisms, [The kinetics of oligonucleotidereplacement. Luis P. Reynaldo, Alexander V. Vologodskii, Bruce P. Neriand Victor I. Lyamichev. J. Mol. Biol. 97: 511-520 (2000)], multipleprobes can hybridize to the same target, allowing multiple cleavages,and the generation of multiple cleavage products.

By the extent of its complementarity to a target nucleic acid strand, anoligonucleotide may be said to define a specific region of said target.In an invasive cleavage structure, the two oligonucleotides define andhybridize to regions of the target that are adjacent to one another(i.e., regions without any additional region of the target betweenthem). Either or both oligonucleotides may comprise additional portionsthat are not complementary to the target strand. In addition tohybridizing adjacently, in order to form an invasive cleavage structure,the 3′ end of the upstream oligonucleotide must comprise an additionalmoiety. When both oligonucleotides are hybridized to a target strand toform a structure and such a 3′ moiety is present on the upstreamoligonucleotide within the structure, the oligonucleotides may be saidto overlap, and the structure may be described as an overlapping, orinvasive cleavage structure.

In one embodiment, the 3′ moiety of the invasive cleavage structure is asingle nucleotide. In this embodiment the 3′ moiety may be anynucleotide (i.e., it may be, but it need not be complementary to thetarget strand). In a preferred embodiment the 3′ moiety is a singlenucleotide that is not complementary to the target strand. In anotherembodiment, the 3′ moiety is a nucleotide-like compound (i.e., a moietyhaving chemical features similar to a nucleotide, such as a nucleotideanalog or an organic ring compound; See e.g., U.S. Pat. No. 5,985,557).In yet another embodiment the 3′ moiety is one or more nucleotides thatduplicate in sequence one or more nucleotides present at the 5′ end ofthe hybridized region of the downstream oligonucleotide. In a furtherembodiment, the duplicated sequence of nucleotides of the 3′ moiety isfollowed by a single nucleotide that is not further duplicative of thedownstream oligonucleotide sequence, and that may be any othernucleotide. In yet another embodiment, the duplicated sequence ofnucleotides of the 3′ moiety is followed by a nucleotide-like compound,as described above.

The downstream oligonucleotide may have, but need not have, additionalmoieties attached to either end of the region that hybridizes to thetarget nucleic acid strand. In a preferred embodiment, the downstreamoligonucleotide comprises a moiety at its 5′ end (i.e., a 5′ moiety). Ina particularly preferred embodiment, said 5′ moiety is a 5′ flap or armcomprising a sequence of nucleotides that is not complementary to thetarget nucleic acid strand.

When an overlapping cleavage structure is formed, it can be recognizedand cleaved by a nuclease that is specific for this structure (i.e., anuclease that will cleave one or more of the nucleic acids in theoverlapping structure based on recognition of this structure, ratherthan on recognition of a nucleotide sequence of any of the nucleic acidsforming the structure). Such a nuclease may be termed a“structure-specific nuclease”. In some embodiments, thestructure-specific nuclease is a 5′ nuclease. In a preferred embodiment,the structure-specific nuclease is the 5′ nuclease of a DNA polymerase.In another preferred embodiment, the DNA polymerase having the 5′nuclease is synthesis-deficient. In another preferred embodiment, the 5′nuclease is a FEN-1 endonuclease. In a particularly preferredembodiment, the 5′ nuclease is thermostable.

In some embodiments, said structure-specific nuclease preferentiallycleaves the downstream oligonucleotide. In a preferred embodiment, thedownstream oligonucleotide is cleaved one nucleotide into the 5′ end ofthe region that is hybridized to the target within the overlappingstructure. Cleavage of the overlapping structure at any location by astructure-specific nuclease produces one or more released portions orfragments of nucleic acid, termed “cleavage products”.

In some embodiments, cleavage of an overlapping structure is performedunder conditions wherein one or more of the nucleic acids in thestructure can disassociate (i.e. un-hybridize, or melt) from thestructure. In one embodiment, full or partial disassociation of a firstcleavage structure allows the target nucleic acid to participate in theformation of one or more additional overlapping cleavage structures. Ina preferred embodiment, the first cleavage structure is partiallydisassociated. In a particularly preferred embodiment only theoligonucleotide that is cleaved disassociates from the first cleavagestructure, such that it may be replaced by another copy of the sameoligonucleotide. In some embodiments, said disassociation is induced byan increase in temperature, such that one or more oligonucleotides canno longer hybridize to the target strand. In other embodiments, saiddisassociation occurs because cleavage of an oligonucleotide producesonly cleavage products that cannot bind to the target strand under theconditions of the reaction. In a preferred embodiment, conditions areselected wherein an oligonucleotide may associate with (i.e., hybridizeto) and disassociate from a target strand regardless of cleavage, andwherein the oligonucleotide may be cleaved when it is hybridized to thetarget as part of an overlapping cleavage structure. In a particularlypreferred embodiment, conditions are selected such that the number ofcopies of the oligonucleotide that can be cleaved when part of anoverlapping structure exceeds the number of copies of the target nucleicacid strand by a sufficient amount that when the first cleavagestructure disassociates, the probability that the target strand willassociate with an intact copy of the oligonucleotide is greater than theprobability that that it will associate with a cleaved copy of theoligonucleotide.

In some embodiments, cleavage is performed by a structure-specificnuclease that can recognize and cleave structures that do not have anoverlap. In a preferred embodiment, cleavage is performed by astructure-specific nuclease having a lower rate of cleavage of nucleicacid structures that do not comprise an overlap, compared to the rate ofcleavage of structures comprising an overlap. In a particularlypreferred embodiment, cleavage is performed by a structure-specificnuclease having less than 1% of the rate of cleavage of nucleic acidstructures that do not comprise an overlap, compared to the rate ofcleavage of structures comprising an overlap.

In some embodiments it is desirable to detect the cleavage of theoverlapping cleavage structure. Detection may be by analysis of cleavageproducts or by analysis of one or more of the remaining uncleavednucleic acids. For convenience, the following discussion will refer tothe analysis of cleavage products, but it will be appreciated by thoseskilled in the art that these methods may as easily be applied toanalysis of the uncleaved nucleic acids in an invasive cleavagereaction. Any method known in the art for analysis of nucleic acids,nucleic acid fragments or oligonucleotides may be applied to thedetection of cleavage products.

In one embodiment, the cleavage products may be identified by chemicalcontent, e.g., the relative amounts of each atom, each particular typeof reactive group or each nucleotide base (Chargaff et al., J. Biol.Chem. 177: 405 [1949]) they contain. In this way, a cleavage product maybe distinguished from a longer nucleic acid from which it was releasedby cleavage, or from other nucleic acids.

In another embodiment, the cleavage products may be distinguished by aparticular physical attribute, including but not limited to length,mass, charge, or charge-to-mass ratio. In yet another embodiment, thecleavage product may be distinguished by a behavior that is related to aphysical attribute, including but not limited to rate of rotation insolution, rate of migration during electrophoresis, coefficient ofsedimentation in centrifugation, time of flight in MALDI-TOF massspectrometry, migration rate or other behavior in chromatography,melting temperature from a complementary nucleic acid, orprecipitability from solution.

Detection of the cleavage products may be through release of a label.Such labels may include, but are not limited to one or more of any ofdyes, radiolabels such as ³²P or ³⁵S, binding moieties such as biotin,mass tags, such as metal ions or chemical groups, charge tags, such aspolyamines or charged dyes, haptens such as digoxgenin, luminogenic,phosphorescent or fluorogenic moieties, and fluorescent dyes, eitheralone or in combination with moieties that can suppress or shiftemission spectra, such as by fluorescence resonance energy transfer(FRET) or collisional fluorescence energy transfer.

In some embodiments, analysis of cleavage products may include physicalresolution or separation, for example by electrophoresis, hybridizationor by selective binding to a support, or by mass spectrometry methodssuch as MALDI-TOF. In other embodiments, the analysis may be performedwithout any physical resolution or separation, such as by detection ofcleavage-induced changes in fluorescence as in FRET-based analysis, orby cleavage-induced changes in the rotation rate of a nucleic acid insolution as in fluorescence polarization analysis.

Cleavage products can be used subsequently in any reaction or read-outmethod that can make use of oligonucleotides. Such reactions include,but are not limited to, modification reactions, such as ligation,tailing with a template-independent nucleic acid polymerase and primerextension with a template-dependent nucleic acid polymerase. Themodification of the cleavage products may be for purposes including, butnot limited to, addition of one or more labels or binding moieties,alteration of mass, addition of specific sequences, or for any otherpurpose that would facilitate analysis of either the cleavage productsor analysis of any other by-product, result or consequence of thecleavage reaction.

Analysis of the cleavage products may involve subsequent steps orreactions that do not modify the cleavage products themselves. Forexample, cleavage products may be used to complete a functionalstructure, such as a competent promoter for in vitro transcription oranother protein binding site. Analysis may include the step of using thecompleted structure for or to perform its function. One or more cleavageproducts may also be used to complete an overlapping cleavage structure,thereby enabling a subsequent cleavage reaction, the products of whichmay be detected or used by any of the methods described herein,including the participation in further cleavage reactions.

Certain preferred embodiments of the invasive cleavage reactions areprovided in the following descriptions. As exemplified by the diagram inFIG. 29, the methods of the present invention employ at least a pair ofoligonucleotides that interact with a target nucleic acid to form acleavage structure for a structure-specific nuclease. In someembodiments, the cleavage structure comprises i) a target nucleic acidthat may be either single-stranded or double-stranded (when adouble-stranded target nucleic acid is employed, it may be renderedsingle stranded, e.g., by heating); ii) a first oligonucleotide, termedthe “probe,” that defines a first region of the target nucleic acidsequence by being the complement of that region (regions X and Z of thetarget as shown in FIG. 29); iii) a second oligonucleotide, termed the“INVADER,” the 5′ part of which defines a second region of the sametarget nucleic acid sequence (regions Y and X in FIG. 29), adjacent toand downstream of the first target region (regions X and Z), and thesecond part of which overlaps into the region defined by the firstoligonucleotide (region X depicts the region of overlap). The resultingstructure is diagrammed in FIG. 29.

While not limiting the invention or the instant discussion to anyparticular mechanism of action, the diagram in FIG. 29 represents theeffect on the site of cleavage caused by this type of arrangement of apair of oligonucleotides. The design of such a pair of oligonucleotidesis described below in detail. In FIG. 29, the 3′ ends of the nucleicacids (i.e., the target and the oligonucleotides) are indicated by theuse of the arrowheads on the ends of the lines depicting the strands ofthe nucleic acids (and where space permits, these ends are also labeled“3′”). It is readily appreciated that the two oligonucleotides (theINVADER and the probe) are arranged in a parallel orientation relativeto one another, while the target nucleic acid strand is arranged in ananti-parallel orientation relative to the two oligonucleotides. Further,it is clear that the INVADER oligonucleotide is located upstream of theprobe oligonucleotide and that with respect to the target nucleic acidstrand, region Z is upstream of region X and region X is upstream ofregion Y (that is, region Y is downstream of region X and region X isdownstream of region Z). Regions of complementarity between the opposingstrands are indicated by the short vertical lines. While not intended toindicate the precise location of the site(s) of cleavage, the area towhich the site of cleavage within the probe oligonucleotide is shiftedby the presence of the INVADER oligonucleotide in this embodiment isindicated by the solid vertical arrowhead. An alternative representationof the target/INVADER/probe cleavage structure is shown in FIG. 32 c.Neither diagram (i.e., FIG. 29 or FIG. 32 c) is intended to representthe actual mechanism of action or physical arrangement of the cleavagestructure and further it is not intended that the method of the presentinvention be limited to any particular mechanism of action.

It can be considered that the binding of these oligonucleotides in thisembodiment divides the target nucleic acid into three distinct regions:one region that has complementarity to only the probe (shown as “Z”);one region that has complementarity only to the INVADER oligonucleotide(shown as “Y”); and one region that has complementarity to botholigonucleotides (shown as “X”). As discussed above, in some preferredembodiments of the present invention, the overlap may comprise moietiesother than overlapping complementary bases. Thus, in some embodiments,the region shown as “X” can represent a region where there is aphysical, but not sequence, overlap between the INVADER and probeoligonucleotides, i.e., in these latter embodiments, there is not aregion of the target nucleic acid between regions “Z” and “Y” that hascomplementarity to both oligonucleotides.

a) Oligonucleotide Design

Design of these oligonucleotides (i.e., the INVADER oligonucleotide andthe probe) is accomplished using practices that are standard in the art.For example, sequences that have self complementarity, such that theresulting oligonucleotides would either fold upon themselves, orhybridize to each other at the expense of binding to the target nucleicacid, are generally avoided.

One consideration in choosing a length for these oligonucleotides is thecomplexity of the sample containing the target nucleic acid. Forexample, the human genome is approximately 3×10⁹ basepairs in length.Any 10-nucleotide sequence will appear with a frequency of 1:4¹⁰, or1:1048,576 in a random string of nucleotides, which would beapproximately 2,861 times in 3 billion basepairs. Clearly, anoligonucleotide of this length would have a poor chance of bindinguniquely to a 10 nucleotide region within a target having a sequence thesize of the human genome. If the target sequence were within a 3 kbplasmid, however, such an oligonucleotide might have a very reasonablechance of binding uniquely. By this same calculation it can be seen thatan oligonucleotide of 16 nucleotides (i.e., a 16-mer) is the minimumlength of a sequence that is mathematically likely to appear once in3×10⁹ basepairs. This level of specificity may also be provided by twoor more shorter oligonucleotides if they are configured to bind in acooperative fashion (i.e., such that they can produce the intendedcomplex only if both or all are bound to their intended targetsequences), wherein the combination of the short oligonucleotidesprovides the desired specificity. In one such embodiment, thecooperativity between the shorter oligonucleotides is by a coaxialstacking effect that can occur when the oligonucleotides hybridize toadjacent sites on a target nucleic acid. In another embodiment, theshorter oligonucleotides are connected to one another, either directly,or by one or more spacer regions. The short oligonucleotides thusconnected may bind to distal regions of the target and may be used tobridge across regions of secondary structure in a target. Examples ofsuch bridging oligonucleotides are described in PCT Publication WO98/50403, herein incorporated by reference in its entirety.

A second consideration in choosing oligonucleotide length is thetemperature range in which the oligonucleotides will be expected tofunction. A 16-mer of average base content (50% G-C bases) will have acalculated T_(m) of about 41° C., depending on, among other things, theconcentration of the oligonucleotide and its target, the salt content ofthe reaction and the precise order of the nucleotides. As a practicalmatter, longer oligonucleotides are usually chosen to enhance thespecificity of hybridization. Oligonucleotides 20 to 25 nucleotides inlength are often used, as they are highly likely to be specific if usedin reactions conducted at temperatures which are near their T_(m)s(within about 5° C. of the T_(m)). In addition, with calculated T_(m)sin the range of 50 to 70° C., such oligonucleotides (i.e., 20 to25-mers) are appropriately used in reactions catalyzed by thermostableenzymes, which often display optimal activity near this temperaturerange.

The maximum length of the oligonucleotide chosen is also based on thedesired specificity. One must avoid choosing sequences that are so longthat they are either at a high risk of binding stably to partialcomplements, or that they cannot easily be dislodged when desired (e.g.,failure to disassociate from the target once cleavage has occurred orfailure to disassociate at a reaction temperature suitable for theenzymes and other materials in the reaction).

The first step of design and selection of the oligonucleotides for theINVADER oligonucleotide-directed cleavage is in accordance with thesesample general principles. Considered as sequence-specific probesindividually, each oligonucleotide may be selected according to theguidelines listed above. That is to say, each oligonucleotide willgenerally be long enough to be reasonably expected to hybridize only tothe intended target sequence within a complex sample, usually in the 20to 40 nucleotide range. Alternatively, because the INVADERoligonucleotide-directed cleavage assay depends upon the concertedaction of these oligonucleotides, the composite length of the 2oligonucleotides which span/bind to the X, Y, Z regions may be selectedto fall within this range, with each of the individual oligonucleotidesbeing in approximately the 13 to 17 nucleotide range. Such a designmight be employed if a non-thermostable cleavage means were employed inthe reaction, requiring the reactions to be conducted at a lowertemperature than that used when thermostable cleavage means areemployed. In some embodiments, it may be desirable to have theseoligonucleotides bind multiple times within a single target nucleic acid(e.g., to bind to multiple variants or multiple similar sequences withina target). It is not intended that the method of the present inventionbe limited to any particular size of the probe or INVADERoligonucleotide.

The second step of designing an oligonucleotide pair for this assay isto choose the degree to which the upstream “INVADER” oligonucleotidesequence will overlap into the downstream “probe” oligonucleotidesequence, and consequently, the sizes into which the probe will becleaved. A key feature of this assay is that the probe oligonucleotidecan be made to “turn over,” that is to say probe can be made to departto allow the binding and cleavage of other copies of the probe molecule,without the requirements of thermal denaturation or displacement bypolymerization. While in one embodiment of this assay probe turnover maybe facilitated by an exonucleolytic digestion by the cleavage agent, itis central to the present invention that the turnover does not requirethis exonucleolytic activity. For example, in some embodiments, areaction temperature and reaction conditions are selected so as tocreate an equilibrium wherein the probe hybridizes and disassociatesfrom the target. In other embodiments, temperature and reactionconditions are selected so that unbound probe can initiate binding tothe target strand and physically displace bound probe. In still otherembodiments, temperature and reaction conditions are selected such thateither or both mechanisms of probe replacement may occur in anyproportion. The method of the present invention is not limited to anyparticular mechanism of probe replacement. By any mechanism, when theprobe is bound to the target to form a cleavage structure, cleavage canoccur. The continuous cycling of the probe on and off of the targetallows multiple probes to bind and be cleaved for each copy of a targetnucleic acid.

i) Choosing the Amount of Sequence Overlap

One way of accomplishing such turnover, where the INVADERoligonucleotide and probe oligonucleotide share a region ofcomplementarity, can be envisioned by considering the diagram in FIG.29. It can be seen that the T_(m) of each oligonucleotide will be afunction of the full length of that oligonucleotide: i.e., the T_(m) ofthe INVADER oligonucleotide=T_(m)(Y+X), and the T_(m) of theprobe=T_(m)(X+Y) for the probe. When the probe is cleaved the X regionis released, leaving the Z section. If the T_(m) of Z is less than thereaction temperature, and the reaction temperature is less than theT_(m)(X+Z), then cleavage of the probe will lead to the departure of Z,thus allowing a new (X+Z) to hybridize. It can be seen from this examplethat the X region must be sufficiently long that the release of X willdrop the T_(m) of the remaining probe section below the reactiontemperature: a G-C rich X section may be much shorter than an A-T rich Xsection and still accomplish this stability shift.

In other embodiments described herein, probe turn over is not related toa change in T_(m) caused by cleavage of the probe, but rather is relatedto the association and disassociation behavior of the probe in theselected conditions, regardless of cleavage. Thus, it is not intendedthat the present invention be limited to the use of probes that, uponcleavage, yield products having a T_(m)s below the reaction temperature,as described above.

ii) Non-Sequence Overlaps

It has been determined that the relationship between the 3′ end of theupstream oligonucleotide and the desired site of cleavage on the probeshould be carefully designed. It is known that the preferred site ofcleavage for the types of structure-specific endonucleases employedherein is one basepair into a duplex (Lyamichev et al., supra). It waspreviously believed that the presence of an upstream oligonucleotide orprimer allowed the cleavage site to be shifted away from this preferredsite, into the single stranded region of the 5′ arm (Lyamichev et al.,supra and U.S. Pat. No. 5,422,253). In contrast to this previouslyproposed mechanism, and while not limiting the present invention to anyparticular mechanism, it is believed that the nucleotide immediately 5′,or upstream of the cleavage site on the probe (including miniprobe andmid-range probes) should be able to basepair with the target forefficient cleavage to occur. In the case of the present invention, thiswould be the nucleotide in the probe sequence immediately upstream ofthe intended cleavage site. In addition, as described herein, it hasbeen observed that in order to direct cleavage to that same site in theprobe, the upstream oligonucleotide should have its 3′ base (i.e., nt)immediately upstream of the intended cleavage site of the probe. Inembodiments where the INVADER and probe oligonucleotides share asequence overlap, this places the 3′ terminal nucleotide of the upstreamoligonucleotide and the base of the probe oligonucleotide 5′ of thecleavage site in competition for pairing with the correspondingnucleotide of the target strand.

To examine the outcome of this competition (i.e. which base is pairedduring a successful cleavage event), substitutions were made in theprobe and INVADER oligonucleotides such that either the probe or theINVADER oligonucleotide were mismatched with the target sequence at thisposition. The effects of both arrangements on the rates of cleavage wereexamined. When the INVADER oligonucleotide is unpaired at the 3′ end,the rate of cleavage was not reduced. If this base was removed, however,the cleavage site was shifted upstream of the intended site. Incontrast, if the probe oligonucleotide was not base-paired to the targetjust upstream of the site to which the INVADER oligonucleotide wasdirecting cleavage, the rate of cleavage was dramatically reduced,suggesting that when a competition exists, the probe oligonucleotide wasthe molecule to be base-paired in this position.

It appears that the 3′ end of the upstream INVADER oligonucleotide isunpaired during cleavage, and yet is important for accurate positioningof the cleavage. To examine which part(s) of the 3′ terminal nucleotideare required for the positioning of cleavage, INVADER oligonucleotideswere designed that terminated on this end with nucleotides that werealtered in a variety of ways. Sugars examined included 2′ deoxyribosewith a 3′ phosphate group, a dideoxyribose, 3′ deoxyribose, 2′ O-methylribose, arabinose and arabinose with a 3′ phosphate. Abasic ribose, withand without 3′ phosphate were tested. Synthetic “universal” bases suchat 3-nitropyrrole and 5-3 nitroindole on ribose sugars were tested.Finally, a base-like aromatic ring structure, acridine, linked to the 3′end the previous nucleotide without a sugar group was tested. Theresults obtained support the conclusion that the aromatic ring of thebase (at the 3′ end of the INVADER oligonuceotide) is an importantmoiety for accomplishing the direction of cleavage to the desired sitewithin the downstream probe. The 3′ terminal moiety of the INVADERoligonucleotide need not be a base that is complementary to the targetnucleic acid.

iii) Miniprobes and Mid-Range Probes;

As discussed above, the INVADER oligonucleotide-directed cleavage assaymay be performed using INVADER and probe oligonucleotides that have alength of about 13-25 nucleotides (typically 20-25 nucleotides). It isalso contemplated that the oligonucleotides may themselves be composedof shorter oligonucleotide sequences that align along a target strandbut that are not covalently linked. This is to say that there is a nickin the sugar-phosphate backbone of the composite oligonucleotide, butthat there is no disruption in the progression of base-pairednucleotides in the resulting duplex. When short strands of nucleic acidalign contiguously along a longer strand the hybridization of each isstabilized by the hybridization of the neighboring fragments because thebasepairs can stack along the helix as though the backbone was in factuninterrupted. This cooperativity of binding can give each segment astability of interaction in excess of what would be expected for thesegment hybridizing to the longer nucleic acid alone. One application ofthis observation has been to assemble primers for DNA sequencing,typically about 18 nucleotides long, from sets of three hexameroligonucleotides that are designed to hybridize in this way (Kotler etal. Proc. Natl. Acad. Sci. USA 90:4241 [1993]). The resultingdoubly-nicked primer can be extended enzymatically in reactionsperformed at temperatures that might be expected to disrupt thehybridization of hexamers, but not of 18-mers.

The use of composite or split oligonucleotides is applied with successin the INVADER-directed cleavage assay. For example, the probeoligonucleotide may be split into two oligonucleotides that anneal in acontiguous and adjacent manner along a target oligonucleotide asdiagrammed in FIG. 57. In this Figure, the downstream oligonucleotide(analogous to the probe of FIG. 25) is assembled from two smallerpieces: a short segment of 6-10 nts (termed the “miniprobe”), that is tobe cleaved in the course of the detection reaction, and anoligonucleotide that hybridizes immediately downstream of the miniprobe(termed the “stacker”), that serves to stabilize the hybridization ofthe probe. To form the cleavage structure, an upstream oligonucleotide(the INVADER oligonucleotide) is provided to direct the cleavageactivity to the desired region of the miniprobe. Assembly of the probefrom non-linked pieces of nucleic acid (i.e., the miniprobe and thestacker) allows regions of sequences to be changed without requiring there-synthesis of the entire proven sequence, thus improving the cost andflexibility of the detection system. In addition, the use of unlinkedcomposite oligonucleotides makes the system more stringent in itsrequirement of perfectly matched hybridization to achieve signalgeneration, allowing this to be used as a sensitive means of detectingmutations or changes in the target nucleic acid sequences.

As illustrated in FIG. 57, in one embodiment, the methods of the presentinvention employ at least three oligonucleotides that interact with atarget nucleic acid to form a cleavage structure for astructure-specific nuclease. More specifically, the cleavage structurecomprises i) a target nucleic acid that may be either single-stranded ordouble-stranded (when a double-stranded target nucleic acid is employed,it may be rendered single-stranded, e.g., by heating); ii) a firstoligonucleotide, termed the “stacker,” that defines a first region ofthe target nucleic acid sequence by being the complement of that region(region W of the target as shown in FIG. 57); iii) a secondoligonucleotide, termed the “miniprobe,” that defines a second region ofthe target nucleic acid sequence by being the complement of that region(regions X and Z of the target as shown in FIG. 57); iv) a thirdoligonucleotide, termed the “INVADER,” the 5′ part of which defines athird region of the same target nucleic acid sequence (regions Y and Xin FIG. 57), adjacent to and downstream of the second target region(regions X and Z), and the second or 3′ part of which overlaps into theregion defined by the second oligonucleotide (region X depicts theregion of overlap). The resulting structure is diagrammed in FIG. 57. Asdescribed above for embodiments that do not employ a stacker, the regionshown as “X” can represent a region where there is a physical, but notsequence, overlap between the INVADER and probe oligonucleotides.

While not limiting the invention or the instant discussion to anyparticular mechanism of action, the diagram in FIG. 57 represents theeffect on the site of cleavage caused by this type of arrangement ofthree oligonucleotides. The design of these three oligonucleotides isdescribed below in detail. In FIG. 57, the 3′ ends of the nucleic acids(i.e., the target and the oligonucleotides) are indicated by the use ofthe arrowheads on the ends of the lines depicting the strands of thenucleic acids (and where space permits, these ends are also labeled“3′”). It is readily appreciated that the three oligonucleotides (theINVADER, the miniprobe and the stacker) are arranged in a parallelorientation relative to one another, while the target nucleic acidstrand is arranged in an anti-parallel orientation relative to the threeoligonucleotides. Further it is clear that the INVADER oligonucleotideis located upstream of the miniprobe oligonucleotide and that theminiprobe olignuceotide is located upstream of the stackeroligonucleotide and that with respect to the target nucleic acid strand,region W is upstream of region Z, region Z is upstream of upstream ofregion X and region X is upstream of region Y (that is region Y isdownstream of region X, region X is downstream of region Z and region Zis downstream of region W). Regions of complementarity between theopposing strands are indicated by the short vertical lines. While notintended to indicate the precise location of the site(s) of cleavage,the area to which the site of cleavage within the miniprobeoligonucleotide is shifted by the presence of the INVADERoligonucleotide is indicated by the solid vertical arrowhead. FIG. 57 isnot intended to represent the actual mechanism of action or physicalarrangement of the cleavage structure and further it is not intendedthat the method of the present invention be limited to any particularmechanism of action.

It can be considered that the binding of these oligonucleotides dividesthe target nucleic acid into four distinct regions: one region that hascomplementarity to only the stacker (shown as “W”); one region that hascomplementarity to only the miniprobe (shown as “Z”); one region thathas complementarity only to the INVADER oligonucleotide (shown as “Y”);and one region that has complementarity to both the INVADER andminiprobe oligonucleotides (shown as “X”). As discussed above, theINVADER oligonucleotide may also be employed such that a physicaloverlap rather than a sequence overlap with the probe is provided.

In addition to the benefits cited above, the use of a composite designfor the oligonucleotides that form the cleavage structure allows morelatitude in the design of the reaction conditions for performing theINVADER-directed cleavage assay. When a longer probe (e.g., 16-25 nt),as described above, is used for detection in reactions that areperformed at temperatures below the T_(m) of that probe, the cleavage ofthe probe may play a significant role in destabilizing the duplex ofwhich it is a part, thus allowing turnover and reuse of the recognitionsite on the target nucleic acid. In contrast, reaction temperatures thatare at or above the T_(m) of the probe mean that the probe molecules arehybridizing and releasing from the target quite rapidly even withoutcleavage of the probe. When an upstream INVADER oligonucleotide and acleavage means are provided the probe will be specifically cleaved, butthe cleavage will not be necessary to the turnover of the probe. When along probe (e.g., 16-25 nt) is used in this way the temperaturesrequired to achieve this state is high, around 65 to 70° C. for a 25-merof average base composition. Requiring the use of such elevatedtemperatures limits the choice of cleavage agents to those that are verythermostable, and may contribute to background in the reactions,depending of the means of detection, through thermal degradation of theprobe oligonucleotides. With miniprobes, this latter mechanism of probereplacement may be accomplished at a lower temperature. Thus, shorterprobes are preferred for embodiments using lower reaction temperatures.

The miniprobe of the present invention may vary in size depending on thedesired application. In one embodiment, the probe may be relativelyshort compared to a standard probe (e.g., 16-25 nt), in the range of 6to 10 nucleotides. When such a short probe is used, reaction conditionscan be chosen that prevent hybridization of the miniprobe in the absenceof the stacker oligonucleotide. In this way a short probe can be made toassume the statistical specificity and selectivity of a longer sequence.In the event of a perturbation in the cooperative binding of theminiprobe and stacker nucleic acids, as might be caused by a mismatchwithin the short sequence that is otherwise complementary to the targetnucleic acid or at the junction between the contiguous duplexes, thiscooperativity can be lost, dramatically reducing the stability of theshorter duplex (i.e., that of the miniprobe), and thus reducing thelevel of cleaved product in the assay of the present invention.

It is also contemplated that probes of intermediate size may be used.Such probes, in the 11 to 15 nucleotide range, may blend some of thefeatures associated with the longer probes as originally described,these features including the ability to hybridize and be cleaved absentthe help of a stacker oligonucleotide. At temperatures below theexpected T_(m) of such probes, the mechanisms of turnover may be asdiscussed above for probes in the 20 nt range, and be dependent on theremoval of the sequence in the ‘X’ region for destabilization andcycling.

The mid-range probes may also be used at elevated temperatures, at orabove their expected T_(m), to allow melting rather than cleavage topromote probe turnover. In contrast to the longer probes describedabove, however, the temperatures required to allow the use of such athermally driven turnover are much lower (about 40 to 60° C.), thuspreserving both the cleavage means and the nucleic acids in the reactionfrom thermal degradation. In this way, the mid-range probes may performin some instances like the miniprobes described above. In a furthersimilarity to the miniprobes, the accumulation of cleavage signal from amid-range probe may be helped under some reaction conditions by thepresence of a stacker.

To summarize, a standard long probe usually does not benefit from thepresence of a stacker oligonucleotide downstream (the exception beingcases where such an oligonucleotide may also disrupt structures in thetarget nucleic acid that interfere with the probe binding), and it maybe used in conditions requiring several nucleotides to be removed toallow the oligonucleotide to release from the target efficiently. Iftemperature of the reaction is used to drive exchange of the probes,standard probes may require use of a temperature at which nucleic acidsand enzymes are at higher risk of thermal degradation.

The miniprobe is very short and performs optimally in the presence of adownstream stacker oligonucleotide. The miniprobes are well suited toreactions conditions that use the temperature of the reaction to driverapid exchange of the probes on the target regardless of whether anybases have been cleaved. In reactions with sufficient amount of thecleavage means, the probes that do bind will be rapidly cleaved beforethey melt off.

The mid-range or midiprobe combines features of these probes and can beused in reactions like those favored by long probes, with longer regionsof overlap (“X” regions) to drive probe turnover at lower temperature.In a preferred embodiment, the midrange probes are used at temperaturessufficiently high that the probes are hybridizing to the target andreleasing rapidly regardless of cleavage. The mid-range probe may haveenhanced performance in the presence of a stacker under somecircumstances.

The distinctions between the mini-, midi- (i.e., mid-range) and longprobes are not contemplated to be inflexible and based only on length.The performance of any given probe may vary with its specific sequence,the choice of solution conditions, the choice of temperature and theselected cleavage means.

It is shown in Example 17 that the assemblage of oligonucleotides thatcomprises the cleavage structure of the present invention is sensitiveto mismatches between the probe and the target. The site of the mismatchused in Ex. 17 provides one example and is not intended to be alimitation in location of a mismatch affecting cleavage. It is alsocontemplated that a mismatch between the INVADER oligonucleotide and thetarget may be used to distinguish related target sequences. In the3-oligonucleotide system, comprising an INVADER, a probe and a stackeroligonucleotide, it is contemplated that mismatches may be locatedwithin any of the regions of duplex formed between theseoligonucleotides and the target sequence. In a preferred embodiment, amismatch to be detected is located in the probe. In a particularlypreferred embodiment, the mismatch is in the probe, at the basepairimmediately upstream (i.e., 5′) of the site that is cleaved when theprobe is not mismatched to the target.

In another preferred embodiment, a mismatch to be detected is locatedwithin the region ‘Z’ defined by the hybridization of a miniprobe. In aparticularly preferred embodiment, the mismatch is in the miniprobe, atthe basepair immediately upstream (i.e., 5′) of the site that is cleavedwhen the miniprobe is not mismatched to the target.

b) Design of the Reaction Conditions

Target nucleic acids that may be analyzed using the methods of thepresent invention that employ a 5′ nuclease or other appropriatecleavage agents include of both RNA and DNA. Such nucleic acids may beobtained using standard molecular biological techniques. For example,nucleic acids (RNA or DNA) may be isolated from a tissue sample (e.g., abiopsy specimen), tissue culture cells, samples containing bacteriaand/or viruses (including cultures of bacteria and/or viruses), etc. Thetarget nucleic acid may also be transcribed in vitro from a DNA templateor may be chemically synthesized or amplified in by polymerase chainreaction. Furthermore, nucleic acids may be isolated from an organism,either as genomic material or as a plasmid or similar extrachromosomalDNA, or they may be a fragment of such material generated by treatmentwith a restriction endonuclease or other cleavage agent, or a shearingforce, or it may be synthetic.

Assembly of the target, probe, and INVADER oligonucleotide nucleic acidsinto the cleavage reaction of the present invention uses principlescommonly used in the design of oligonucleotide-based enzymatic assays,such as dideoxynucleotide sequencing and polymerase chain reaction(PCR). As is done in these assays, the oligonucleotides are provided insufficient excess that the rate of hybridization to the target nucleicacid is very rapid. These assays are commonly performed with 50 fmolesto 2 pmoles of each oligonucleotide per microliter of reaction mixture,although they are not necessarily limited to this range In the Examplesdescribed herein, amounts of oligonucleotides ranging from 250 fmoles to5 pmoles per microliter of reaction volume were used. These values werechosen for the purpose of ease in demonstration and are not intended tolimit the performance of the present invention to these concentrations.Other (e.g., lower) oligonucleotide concentrations commonly used inother molecular biological reactions are also contemplated.

It is desirable that an INVADER oligonucleotide be immediately availableto direct the cleavage of each probe oligonucleotide that hybridizes toa target nucleic acid. In some embodiments described herein, the INVADERoligonucleotide is provided in excess over the probe oligonucleotide.While this is an effective means of making the INVADER oligonucleotideimmediately available in such embodiments it is not intended that thepractice of the present invention be limited to conditions wherein theINVADER oligonucleotide is in excess over the probe, or to anyparticular ratio of INVADER-to-probe (e.g., in some preferredembodiments described herein, the probe is provided in excess over theINVADER oligonucleotide). Another means of assuring the presence of anINVADER oligonucleotide whenever a probe binds to a target nucleic acidis to design the INVADER oligonucleotide to hybridize more stably to thetarget, i.e., to have a higher T_(m) than the probe. This can beaccomplished by any of the means of increasing nucleic acid duplexstability discussed herein (e.g., by increasing the amount ofcomplementarity to the target nucleic acid).

Buffer conditions should be chosen that will be compatible with both theoligonucleotide/target hybridization and with the activity of thecleavage agent. The optimal buffer conditions for nucleic acidmodification enzymes, and particularly DNA modification enzymes,generally included enough mono- and di-valent salts to allow associationof nucleic acid strands by base-pairing. If the method of the presentinvention is performed using an enzymatic cleavage agent other thanthose specifically described here, the reactions may generally beperformed in any such buffer reported to be optimal for the nucleasefunction of the cleavage agent. In general, to test the utility of anycleavage agent in this method, test reactions are performed wherein thecleavage agent of interest is tested in the MOPS/MnCl₂/KCl buffer orMg-containing buffers described herein and in whatever buffer has beenreported to be suitable for use with that agent, in a manufacturer'sdata sheet, a journal article, or in personal communication.

The products of the INVADER oligonucleotide-directed cleavage reactionare fragments generated by structure-specific cleavage of the inputoligonucleotides. The resulting cleaved and/or uncleavedoligonucleotides may be analyzed and resolved by a number of methodsincluding, but not limited to, electrophoresis (on a variety of supportsincluding acrylamide or agarose gels, paper, etc.), chromatography,fluorescence polarization, mass spectrometry and chip hybridization. Insome Examples the invention is illustrated using electrophoreticseparation for the analysis of the products of the cleavage reactions.However, it is noted that the resolution of the cleavage products is notlimited to electrophoresis. Electrophoresis is chosen to illustrate themethod of the invention because electrophoresis is widely practiced inthe art and is easily accessible to the average practitioner. In otherExamples, the invention is illustrated without electrophoresis or anyother resolution of the cleavage products.

The probe and INVADER oligonucleotides may contain a label to aid intheir detection following the cleavage reaction. The label may be aradioisotope (e.g., a ³²P or ³⁵S-labelled nucleotide) placed at eitherthe 5′ or 3′ end of the oligonucleotide or alternatively, the label maybe distributed throughout the oligonucleotide (i.e., a uniformly labeledoligonucleotide). The label may be a nonisotopic detectable moiety, suchas a fluorophore, that can be detected directly, or a reactive groupthat permits specific recognition by a secondary agent. For example,biotinylated oligonucleotides may be detected by probing with astreptavidin molecule that is coupled to an indicator (e.g., alkalinephosphatase or a fluorophore) or a hapten such as dioxigenin may bedetected using a specific antibody coupled to a similar indicator. Thereactive group may also be a specific configuration or sequence ofnucleotides that can bind or otherwise interact with a secondary agent,such as another nucleic acid, and enzyme, or an antibody.

c) Optimization of Reaction Conditions

The INVADER oligonucleotide-directed cleavage reaction is useful todetect the presence of specific nucleic acids. In addition to theconsiderations listed above for the selection and design of the INVADERand probe oligonucleotides, the conditions under which the reaction isto be performed may be optimized for detection of a specific targetsequence.

One objective in optimizing the INVADER oligonucleotide-directedcleavage assay is to allow specific detection of the fewest copies of atarget nucleic acid. To achieve this end, it is desirable that thecombined elements of the reaction interact with the maximum efficiency,so that the rate of the reaction (e.g., the number of cleavage eventsper minute) is maximized. Elements contributing to the overallefficiency of the reaction include the rate of hybridization, the rateof cleavage, and the efficiency of the release of the cleaved probe.

The rate of cleavage will be a function of the cleavage means chosen,and may be made optimal according to the manufacturer's instructionswhen using commercial preparations of enzymes or as described in theexamples herein. The other elements (rate of hybridization, efficiencyof release) depend upon the execution of the reaction, and optimizationof these elements is discussed below.

Three elements of the cleavage reaction that significantly affect therate of nucleic acid hybridization are the concentration of the nucleicacids, the temperature at which the cleavage reaction is performed andthe concentration of salts and/or other charge-shielding ions in thereaction solution.

The concentrations at which oligonucleotide probes are used in assays ofthis type are well known in the art, and are discussed above. Oneexample of a common approach to optimizing an oligonucleotideconcentration is to choose a starting amount of oligonucleotide forpilot tests; 0.01 to 2 μM is a concentration range used in manyoligonucleotide-based assays. When initial cleavage reactions areperformed, the following questions may be asked of the data: Is thereaction performed in the absence of the target nucleic acidsubstantially free of the cleavage product?; Is the site of cleavagespecifically positioned in accordance with the design of the INVADERoligonucleotide?; Is the specific cleavage product easily detected inthe presence of the uncleaved probe (or is the amount of uncut materialoverwhelming the chosen visualization method)?

A negative answer to any of these questions would suggest that the probeconcentration is too high, and that a set of reactions using serialdilutions of the probe should be performed until the appropriate amountis identified. Once identified for a given target nucleic acid in a givesample type (e.g., purified genomic DNA, body fluid extract, lysedbacterial extract), it should not need to be re-optimized. The sampletype is important because the complexity of the material present mayinfluence the probe concentration optimum.

Conversely, if the chosen initial probe concentration is too low, thereaction may be slow, due to inefficient hybridization. Tests withincreasing quantities of the probe will identify the point at which theconcentration exceeds the optimum (e.g., at which it produces anundesirable effect, such as background cleavage not dependent on thetarget sequence, or interference with detection of the cleavedproducts). Since the hybridization will be facilitated by excess ofprobe, it is desirable, but not required, that the reaction be performedusing probe concentrations just below this point.

The concentration of INVADER oligonucleotide can be chosen based on thedesign considerations discussed above. In some embodiments, the INVADERoligonucleotide is in excess of the probe oligonucleotide. In apreferred embodiment, the probe oligonucleotide is in excess of theINVADER oligonucleotide.

Temperature is also an important factor in the hybridization ofoligonucleotides. The range of temperature tested will depend in largepart on the design of the oligonucleotides, as discussed above. Where itis desired to have a reaction be run at a particular temperature (e.g.,because of an enzyme requirement, for convenience, for compatibilitywith assay or detection apparatuses, etc.), the oligonucleotides thatfunction in the reaction can be designed to optimally perform at thedesired reaction temperature. Each INVADER reaction includes at leasttwo target sequence-specific oligonucleotides for the primary reaction:an upstream INVADER oligonucleotide and a downstream probeoligonucleotide. In some preferred embodiments, the INVADERoligonucleotide is designed to bind stabily at the reaction temperature,while the probe is designed to freely associate and disassociate withthe target strand, with cleavage occurring only when an uncut probehybridizes adjacent to an overlapping INVADER oligonucleotide. Inpreferred embodiments, the probe includes a 5′ flap that is notcomplementary to the target, and this flap is released from the probewhen cleavage occurs. The released flap can be detected directly orindirectly. In some preferred embodiments, as discussed in detail below,the released flap participate as in INVADER oligonucleotide in asecondary reaction.

Optimum conditions for the INVADER assay are generally those that allowspecific detection of the smallest amount of a target nucleic acid. Suchconditions may be characterized as those that yield the highesttarget-dependent signal in a given timeframe, or for a given amount oftarget nucleic acid, or that allow the highest rate of probe cleavage(i.e., probes cleaved per minute). To select a probe sequence that willperform optimally at a pre-selected reaction temperature, the meltingtemperature (T_(m)) of its analyte specific region (ASR, the region thatis complementary to the target nucleic acid) is calculated using thenearest-neighbor model and published parameters for DNA duplex formation(SantaLucia, J., Proc Natl Acad Sci USA 95, 1460-5 (1998), Allawi, H. T.& SantaLucia, J., Jr. Biochemistry 36, 10581-94 (1997). However, thereare several differences between the conditions under which the publishedparameters were measured and the conditions under which the INVADERassay is run in preferred embodiments. The salt concentrations are oftendifferent than the solution conditions in which the nearest-neighborparameters were obtained (1M NaCl and no divalent metals). One cancompensate for this factor by varying the value provided for the saltconcentration within the melting temperature calculations. In additionto the salt concentration, the presence of and concentration of theenzyme influences the optimal reaction temperature, and an additionaladjustment should be made to the calculated T_(m) to determine theoptimal temperature at which to perform a reaction. By observing theoptimal temperature for a number of INVADER reactions (i.e., thetemperature at which the rate of signal accumulation is highest) it hasbeen possible to further alter the value for salt concentration withinthese calculations to allow the algorithm for T_(m) calculation to bemodified to instead provide an optimal cleavage reaction temperature fora given probe sequence. This additional adjustment is termed a “saltcorrection”. As used herein, the term “salt correction” refers to avariation made in the value provided for a salt concentration, for thepurpose of reflecting the effect on a T_(m) calculation for a nucleicacid duplex of a non-salt parameter or condition affecting said duplex.Variation of the values provided for the strand concentrations will alsoaffect the outcome of these calculations. By using a value of 0.5 M NaCl[SantaLucia, J., Proc Natl Acad Sci USA 95, 1460-5 (1998)] and strandconcentrations of about 1 μM of the probe and 1 fM target, the algorithmused for calculating probe-target melting temperature has been adaptedfor use in predicting optimal INVADER assay reaction temperature. For aset of about 30 probes, the average deviation between optimal assaytemperatures calculated by this method and those experimentallydetermined was about 1.5° C.

As noted above, the concentration of the cleavage agent can affect theactual optimum temperature for a cleavage reaction. Additionally,different cleavage agents, even if used at identical concentrations, canaffect reaction temperature optima differently (e.g., the differencebetween the calculated probe T_(m) and the observed optimal reactiontemperature may be greater for one enzyme than for another).Determination of appropriate salt corrections for reactions usingdifferent enzymes or concentrations of enzymes, or for any othervariation made in reaction conditions, involves a two step process of a)measuring reaction temperature optima under the new reaction conditions,and varying the salt concentration within the T_(m) algorithm to producea calculated temperature matching or closely approximating the observedoptima. Measurement of an optimum reaction temperature generallyinvolves performing reactions at a range of temperatures selected suchthat the range allows observation of an increase in performance as anoptimal temperature is approached (either by increasing or decreasingtemperatures), and a decrease in performance when an optimal temperaturehas been passed, thereby allowing identification of the optimaltemperature or temperature range [see, for example, V. I. Lyamichev, etal., Biochemistry 39, No. 31: 9523-9532 (2000)].

The length of the downstream probe analyte-specific region (ASR) isdefined by the temperature selected for running the reaction, e.g., 63°C. in the experiments described in Examples 54 through 60. To select aprobe sequence based on a desired reaction temperature, the probesequence is selected in the following way (as illustrated for the designof a probe for the detection of a sequence difference at a particularlocation). Starting from the position of the variant nucleotide on thetarget DNA (position N, FIG. 112); the target base that is paired to theprobe nucleotide 5′ of the intended cleavage site), an iterativeprocedure is used by which the length of the ASR is increased by onebase pair until a calculated optimal reaction temperature (T_(m) plussalt correction to compensate for enzyme and any other reactionconditions effects) matching the desired reaction temperature isreached. The non-complementary arm of the probe is preferably selected(by a similar iterative process) to allow the secondary reaction tocycle at the same reaction temperature, and the entire probe design (ASRand 5′ noncomplementary arm) is screened using programs such as mfold[Zuker, M. Science 244, 48-52 (1989)] or Oligo 5.0 [Rychlik, W. &Rhoads, R. E. Nucleic Acids Res 17, 8543-51 (1989)] for the possibleformation of dimer complexes or secondary structures that couldinterfere with the reaction. The same principles are also followed forINVADER oligonucleotide design. The following describes design of anINVADER assay embodiment wherein the 3′ end of the INVADERoligonucleotide, at position N on the target DNA, is designed to have anucleotide not complementary to either allele suspected of beingcontained in the sample to be tested. The mismatch does not adverselyaffect cleavage [Lyamichev, V. et al. Nature Biotechnology 17, 292-296(1999)], and it can enhance probe cycling, presumably by minimizingcoaxial stabilization effects between the two probes. Briefly, startingfrom the position N, additional residues complementary to the target DNAstarting from residue N-1 are then added in the upstream direction untilthe stability of the INVADER-target hybrid exceeds that of the probe(and therefore the planned assay reaction temperature). In preferredembodiments, the stability of the INVADER-target hybrid exceeds that ofthe probe by 15-20° C.

In some embodiments, where the released cleavage fragment from a primaryreaction is to be used in a secondary reaction, one should also considerthe reaction conditions of the secondary reaction in designing theoligonucleotides for the primary reaction (e.g., the sequence of thereleased non-complementary 5′ flap of the probe in the primary reactioncan be designed to optimally function in a secondary reaction). Forexample, as described in detail below, in some embodiments, a secondaryreaction is used where the released cleavage fragment from a primaryreaction hybridizes to a synthetic cassette to form a secondary cleavagereaction. In some preferred embodiments, the cassette comprises afluorescing moiety and a quenching moiety, wherein cleavage of thesecondary cleavage structure separates the fluorescing moiety from thequenching moiety, resulting in a detectable signal (e.g., FRETdetection). The secondary reaction can be configured a number ofdifferent ways. For example, in some embodiments, the synthetic cassettecomprises two oligonucleotides: an oligonucleotide that contains theFRET moieties and a FRET/INVADER oligonucleotide bridgingoligonucleotide that allows the INVADER oligonucleotide (i.e., thereleased flap from the primary reaction) and the FRET oligonucleotide tohybridize thereto, such that a cleavage structure is formed. In someembodiments, the synthetic cassette is provided as a singleoligonucleotide, comprising a hairpin structure (i.e., the FREToligonucleotide is connected at its 3′ end to the bridgingoligonucleotide by a loop). The loop may be nucleic acid, (e.g., astring of nucleotides, such as the four T residues depicted in severalFigures, including 113A) or a non-nucleic acid spacer or linker. Thelinked molecules may together be described as a FRET cassette. In thesecondary reaction using a FRET cassette the released flap from theprimary reaction, which acts as an INVADER oligonucleotide, should beable to associate and disassociate with the FRET cassette freely, sothat one released flap can direct the cleavage of multiple FRETcassettes. It is one aspect of the assay design that all of the probesequences may be selected to allow the primary and secondary reactionsto occur at the same optimal temperature, so that the reaction steps canrun simultaneously. In an alternative embodiment, the probes may bedesigned to operate at different optimal temperatures, so that thereactions steps are not simultaneously at their temperature optima. Asnoted above, the same iterative process used to select the ASR of theprobe can be used in the design of the portion of the primary probe thatparticipates in a secondary reaction.

Another determinant of hybridization efficiency is the saltconcentration of the reaction. In large part, the choice of solutionconditions will depend on the requirements of the cleavage agent, andfor reagents obtained commercially, the manufacturer's instructions area resource for this information. When developing an assay utilizing anyparticular cleavage agent, the oligonucleotide and temperatureoptimizations described above should be performed in the bufferconditions best suited to that cleavage agent.

A “no enzyme” control allows the assessment of the stability of thelabeled oligonucleotides under particular reaction conditions, or in thepresence of the sample to be tested e.g., in assessing the sample forcontaminating nucleases). In this manner, the substrate andoligonucleotides are placed in a tube containing all reactioncomponents, except the enzyme and treated the same as theenzyme-containing reactions. Other controls may also be included. Forexample, a reaction with all of the components except the target nucleicacid will serve to confirm the dependence of the cleavage on thepresence of the target sequence.

d) Selection of a Cleavage Agent

As demonstrated in a number of the Examples, some 5′ nucleases do notrequire an upstream oligonucleotide to be active in a cleavage reaction.Although cleavage may be slower without the upstream oligonucleotide, itmay still occur (Lyamichev et al., Science 260:778 [1993], Kaiser etal., J. Biol. Chem., 274:21387 [1999]). When a DNA strand is thetemplate or target strand to which probe oligonucleotides arehybridized, the 5′ nucleases derived from DNA polymerases and some flapendonucleases (FENs), such as that from Methanococcus jannaschii, cancleave quite well without an upstream oligonucleotide providing anoverlap (Lyamichev et al., Science 260:778 [1993], Kaiser et al., J.Biol. Chem., 274:21387 [1999], and U.S. Pat. No. 5,843,669, hereinincorporated by reference in its entirety). These nucleases may beselected for use in some embodiments of the INVADER assay, e.g., inembodiments wherein cleavage of th probe in the absence of an INVADERoligonucleotide gives a different cleavage product, which does notinterfere with the intended analysis, or wherein both types of cleavage,INVADER oligonucleotide-directed and INVADERoligonucleotide-independent, are intended to occur.

In other embodiments it is preferred that cleavage of the probe bedependent on the presence of an upstream INVADER oligonucleotide, andenzyme having this requirement would be used. Other FENs, such as thosefrom Archeaoglobus fulgidus (Afu) and Pyrococcus furiosus (Pfu), cleavean overlapped structure on a DNA target at so much greater a rate thanthey do a non-overlapping structure (i.e., either missing the upstreamoligonucleotide or having a non-overlapping upstream oligonucleotide)that they can be viewed as having an essentially absolute requirementfor the overlap (Lyamichev et al., Nat. Biotechnol., 17:292 [1999],Kaiser et al., J. Biol. Chem., 274:21387 [1999]). When an RNA target ishybridized to DNA oligonucleotide probes to form a cleavage structure,many FENs cleave the downstream DNA probe poorly, regardless of thepresence of an overlap. On such an RNA-containing structure, the 5′nucleases derived from DNA polymerases have a strong requirement for theoverlap, and are essentially inactive in its absence.

e) Probing for Multiple Alleles

The INVADER oligonucleotide-directed cleavage reaction is also useful inthe detection and quantification of individual variants or alleles in amixed sample population. By way of example, such a need exists in theanalysis of tumor material for mutations in genes associated withcancers. Biopsy material from a tumor can have a significant complementof normal cells, so it is desirable to detect mutations even whenpresent in fewer than 5% of the copies of the target nucleic acid in asample. In this case, it is also desirable to measure what fraction ofthe population carries the mutation. Similar analyses may also be doneto examine allelic variation in other gene systems, and it is notintended that the method of the present invention by limited to theanalysis of tumors.

As demonstrated below, in one embodiment, reactions can be performedunder conditions that prevent the cleavage of probes bearing even asingle-nucleotide difference mismatch within the region of the targetnucleic acid termed “Z” in FIG. 29, but that permit cleavage of asimilar probe that is completely complementary to the target in thisregion. In a preferred embodiment, a mismatch is positioned at thenucleotide in the probe that is 5′ of the site where cleavage occurs inthe absence of the mismatch.

In other embodiments, the INVADER assay may be performed underconditions that have a tight requirement for an overlap (e.g., using theAfu FEN for DNA target detection or the 5′ nuclease of DNA polymerasefor RNA target detection, as described above), providing an alternativemeans of detecting single nucleotide or other sequence variations. Inone embodiment, the probe is selected such that the target basesuspected of varying is positioned at the 5′ end of thetarget-complementary region of this probe. The upstream INVADERoligonucleotide is positioned to provide a single base of overlap. Ifthe target and the probe oligonucleotide are complementary at the basein question, the overlap forms and cleavage can occur. This embodimentis diagrammed in FIG. 112. However, if the target does not complementthe probe at this position, that base in the probe becomes part of anon-complementary 5′ arm, no overlap between the INVADER oligonucleotideand probe oligonucleotide exists, and cleavage is suppressed.

It is also contemplated that different sequences may be detected in asingle reaction. Probes specific for the different sequences may bedifferently labeled. For example, the probes may have different dyes orother detectable moieties, different lengths, or they may havedifferences in net charges of the products after cleavage. Whendifferently labeled in one of these ways, the contribution of eachspecific target sequence to final product can be tallied. This hasapplication in detecting the quantities of different versions of a genewithin a mixture. Different genes in a mixture to be detected andquantified may be wild type and mutant genes (e.g., as may be found in atumor sample, such as a biopsy). In this embodiment, one might designthe probes to precisely the same site, but one to match the wild-typesequence and one to match the mutant. Quantitative detection of theproducts of cleavage from a reaction performed for a set amount of timewill reveal the ratio of the two genes in the mixture. Such analysis mayalso be performed on unrelated genes in a mixture. This type of analysisis not intended to be limited to two genes. Many variants within amixture may be similarly measured.

Alternatively, different sites on a single gene may be monitored andquantified to verify the measurement of that gene. In this embodiment,the signal from each probe would be expected to be the same.

It is also contemplated that multiple probes may be used that are notdifferently labeled, such that the aggregate signal is measured. Thismay be desirable when using many probes designed to detect a single geneto boost the signal from that gene. This configuration may also be usedfor detecting unrelated sequences within a mix. For example, in bloodbanking it is desirable to know if any one of a host of infectiousagents is present in a sample of blood. Because the blood is discardedregardless of which agent is present, different signals on the probeswould not be required in such an application of the present invention,and may actually be undesirable for reasons of confidentiality.

Just as described for the two-oligonucleotide system, above, thespecificity of the detection reaction will be influenced by theaggregate length of the target nucleic acid sequences involved in thehybridization of the complete set of the detection oligonucleotides. Forexample, there may be applications in which it is desirable to detect asingle region within a complex genome. In such a case the set ofoligonucleotides may be chosen to require accurate recognition byhybridization of a longer segment of a target nucleic acid, often in therange of 20 to 40 nucleotides. In other instances it may be desirable tohave the set of oligonucleotides interact with multiple sites within atarget sample. In these cases one approach would be to use a set ofoligonucleotides that recognize a smaller, and thus statistically morecommon, segment of target nucleic acid sequence.

In one preferred embodiment, the INVADER and stacker oligonucleotidesmay be designed to be maximally stable, so that they will remain boundto the target sequence for extended periods during the reaction. Thismay be accomplished through any one of a number of measures well knownto those skilled in the art, such as adding extra hybridizing sequencesto the length of the oligonucleotide (up to about 50 nts in totallength), or by using residues with reduced negative charge, such asphosphorothioates or peptide-nucleic acid residues, so that thecomplementary strands do not repel each other to degree that naturalstrands do. Such modifications may also serve to make these flankingoligonucleotides resistant to contaminating nucleases, thus furtherensuring their continued presence on the target strand during the courseof the reaction. In addition, the INVADER and stacker oligonucleotidesmay be covalently attached to the target (e.g., through the use ofpsoralen cross-linking).

II. Effect of ARRESTOR Molecules on Signal and Background in SequentialInvasive Cleavage Reactions.

As described above, and demonstrated in Example 36, the concentration ofthe probe that is cleaved can be used to increase the rate of signalaccumulation, with higher concentrations of probe yielding higher finalsignal. However, the presence of large amounts of residual uncleavedprobe can present problems for subsequent use of the cleaved productsfor detection or for further amplification. If the subsequent step is asimple detection (e.g., by gel resolution), the excess uncut materialmay cause background by streaking or scattering of signal, or byoverwhelming a detector (e.g., over-exposing a film in the case ofradioactivity, or exceeding the quantitative detection limits of afluorescence imager). This can be overcome by partitioning the productfrom the uncut probe (e.g., by using the charge reversal methoddescribed in Example 22 and discussed in detail below). In more complexdetection methods, the cleaved product may be intended to interact withanother entity to indicate cleavage. As noted above, the cleaved productcan be used in any reaction that makes use of oligonucleotides, such, ashybridization, primer extension, ligation, or the direction of invasivecleavage. In each of these cases, the fate of the residual uncut probeshould be considered in the design of the reaction. In a primerextension reaction, the uncut probe can hybridize to a template forextension. If cleavage is required to reveal the correct 3′ end forextension, the hybridized uncut probe will not be extended. It may,however, compete with the cleaved product for the template. If thetemplate is in excess of the combination of cleaved and uncleaved probe,then both of the latter should be able to find a copy of template forbinding. If, however, the template is limiting, any competition mayreduce the portion of the cleaved probe that can find successfully bindto the available template. If a vast excess of probe was used to drivethe initial reaction, the remainder may also be in vast excess over thecleavage product, and thus may provide a very effective competitor,thereby reducing the amount of the final reaction (e.g., extension)product for ultimate detection.

The participation of the uncut probe material in a secondary reactioncan also contribute to background in these reactions. While thepresentation of a cleaved probe for a subsequent reaction may representan ideal substrate for the enzyme to be used in the next step, someenzymes may also be able to act, albeit inefficiently, on the uncutprobe as well. It was shown in Example 43 that transcription can bepromoted from a nicked promoter even when one side of the nick hasadditional unpaired nucleotides (termed a “branched promoter” in thatExample). Similarly, when the subsequent reaction is to be an invasivecleavage, the uncleaved probe may bind to the elements intended to formthe second cleavage structure with the cleaved probe. Two of thepossible configurations are shown schematically in FIGS. 105 and 106.The right hand structure in the second step in each Figure shows apossible configuration formed by the secondary reaction elements (e.g.,secondary targets and/or probes) and the uncleaved primary probe. Ineach of these cases, it was found that some of the 5′ nucleasesdescribed herein can catalyze some measure of cleavage of thesedefective structures. Even at a low level, this aberrant cleavage can bemisinterpreted as positive target-specific cleavage signal.

With these negative effects of the surfeit of uncut probe considered,there is clearly a need for some method of preventing theseinteractions. As noted above, it is possible to partition the cleavedproduct from the uncut probe after the primary reaction by traditionalmethods. However, these methods are often time consuming, may beexpensive (e.g., disposable columns, gels, etc.), and may increase therisk for sample mishandling or contamination. It is far preferable toconfigure the sequential reactions such that the original sample neednot be removed to a new vessel for subsequent reaction.

The present invention provides a method for reducing interactionsbetween the primary probe and other reactants. This method provides ameans of specifically diverting the uncleaved probes from participationin the subsequent reactions. The diversion is accomplished by theinclusion in the next reaction step an agent designed to specificallyinteract with the uncleaved primary probe. While the primary probe in aninvasive cleavage reaction is discussed for reasons of convenience, itis contemplated that the ARRESTOR molecules may be used at any reactionstep within a chain of invasive cleavage steps, as needed or desired forthe design of an assay. It is not intended that the ARRESTOR moleculesof the present invention be limited to any particular step.

The method of diverting the residual uncut probes from a primaryreaction makes use of agents that can be specifically designed orselected to bind to the uncleaved probe molecules with greater affinitythan to the cleaved probes, thereby allowing the cleaved probe speciesto effectively compete for the elements of the subsequent reaction, evenwhen the uncut probe is present in vast excess. These agents have beentermed “ARRESTOR molecules,” due to their function of stopping orarresting the primary probe from participation in the later reaction. Invarious Examples below, an oligonucleotide is provided as an ARRESTORoligonucleotide in an invasive cleavage assay. It can be appreciatedthat any molecule or chemical that can discriminate between thefull-length uncut probe and the cleaved probe, and that can bind orotherwise disable the uncleaved probe preferentially may be configuredto act as an ARRESTOR molecules within the meaning of the presentinvention. For example, antibodies can be derived with such specificity,as can the “aptamers” that can be selected through multiple steps of invitro amplification (e.g., “SELEX,” U.S. Pat. Nos. 5,270,163 and5,567,588; herein incorporated by reference) and specific rounds ofcapture or other selection means.

In one embodiment, the ARRESTOR molecule is an oligonucleotide. Inanother embodiment the ARRESTOR oligonucleotides is a compositeoligonucleotide, comprising two or more short oligonucleotides that arenot covalently linked, but that bind cooperatively and are stabilized byco-axial stacking. In a preferred embodiment, the oligonucleotide ismodified to reduce interactions with the cleavage agents of the presentinvention. When an oligonucleotide is used as an ARRESTORoligonucleotide, it is intended that it not participate in thesubsequent reactive step. Consideration of the schematic diagrams inFIGS. 105 and 106, particularly the right-most Figure in step 2b of eachFigure, will show that the binding of the ARRESTOR oligonucleotide tothe primary probe may, either with the participation of the secondarytarget, or without such participation, create a bifurcated structurethat is a substrate for cleavage by the 5′ nucleases used in someembodiments of the methods of the present invention. Formation of suchstructures would lead to some level of unintended cleavage that couldcontribute to background, reduce specific signal or compete for theenzyme. It is preferable to provide ARRESTOR oligonucleotides that willnot create such cleavage structures. One method of doing this is to addto the ARRESTOR oligonucleotides such modifications as have been foundto reduce the activity of INVADER oligonucleotides, as the INVADERoligonucleotides occupy a similar position within a cleavage structure(i.e., the 3′ end of the INVADER oligonucleotide positions the site ofcleavage of an unpaired 5′ arm). Modification of the 3′ end of theINVADER oligonucleotides was examined for the effects on cleavage inExample 35; a number of the modifications tested were found to besignificantly debilitating to the function of the INVADERoligonucleotide. Other modifications not described herein may be easilycharacterized by performing such a test using the cleavage enzyme to beused in the reaction for which the ARRESTOR oligonucleotide is intended.

In a preferred embodiment, the backbone of an ARRESTOR oligonucleotideis modified. This may be done to increase the resistance to degradationby nucleases or temperature, or to provide duplex structure that is aless favorable substrate for the enzyme to be used (e.g., A-form duplexvs. B-form duplex). In particularly preferred embodiment, the backbonemodified oligonucleotide further comprises a 3′ terminal modification.In a preferred embodiment, the modifications comprise 2′ O-methylsubstitution of the nucleic acid backbone, while in a particularlypreferred embodiment, the 2′ O-methyl modified oligonucleotide furthercomprises a 3′ terminal amine group.

The purpose of the ARRESTOR oligonucleotide is to allow the minoritypopulation of cleaved probe to effectively compete with the uncleavedprobe for binding whatever elements are to be used in the next step.While an ARRESTOR oligonucleotide that can discriminate between the twoprobe species absolutely (i.e., binding only to uncut and never to cut)may be of the greatest benefit in some embodiments, it is envisionedthat in many applications, including the sequential INVADER assaysdescribed herein, the ARRESTOR oligonucleotide of the present inventionmay perform the intended function with only partial discrimination. Whenthe ARRESTOR oligonucleotide has some interaction with the cleavedprobe, it may prevent detection of some portion of these cleavageproducts, thereby reducing the absolute level of signal generated from agiven amount of target material. If this same ARRESTOR oligonucleotidehas the simultaneous effect of reducing the background of the reaction(i.e., from non-target specific cleavage) by a factor that is greaterthan the factor of reduction in the specific signal, then thesignificance of the signal (i.e., the ratio of signal to background), isincreased, even with the lower amount of absolute signal. Any potentialARRESTOR molecule design may be tested in a simple fashion by comparingthe levels of background and specific signals from reactions that lackARRESTOR molecules to the levels of background and specific signal fromsimilar reactions that include ARRESTOR oligonucleotides. Each of thereactions described in Examples 49-53 demonstrate the use of suchcomparisons, and these can easily be adapted by those skilled in the artto other ARRESTOR molecules and target embodiments. What constitutes anacceptable level of tradeoff of absolute signal for specificity willvary for different applications (e.g., target levels, read-outsensitivity, etc.), and can be determined by any individual user usingthe methods of the present invention.

III. Signal Enhancement by Incorporating the Products of an InvasiveCleavage Reaction Into a Subsequent Invasive Cleavage Reaction

As noted above, the oligonucleotide product released by the invasivecleavage can be used subsequently in any reaction or read-out methodthat uses oligonucleotides in the size range of a cleavage product. Inaddition to the reactions involving primer extension and transcription,described herein, another enzymatic reaction that makes use ofoligonucleotides is the invasive cleavage reaction. The presentinvention provide means of using the oligonucleotide released in aprimary invasive cleavage reaction as a component to complete a cleavagestructure to enable a secondary invasive cleavage reaction. One possibleconfiguration of a primary cleavage reaction supplying a component for asecondary cleavage structure is diagrammed in FIG. 96. Is not intendedthat the sequential use of the invasive cleavage product be limited to asingle additional step. It is contemplated that many distinct invasivecleavage reactions may be performed in sequence.

The polymerase chain reaction uses a DNA replication method to createcopies of a targeted segment of nucleic acid at a logarithmic rate ofaccumulation. This is made possible by the fact that when the strands ofDNA are separated, each individual strand contains sufficientinformation to allow assembly of a new complementary strand. When thenew strands are synthesized the number of identical molecules hasdoubled. Within 20 iterations of this process, the original may becopied 1 million-fold, making very rare sequences easily detectable. Themathematical power of a doubling reaction has been incorporated into anumber of amplification assays, several of which are cited in Table 1.

By performing multiple, sequential invasive cleavage reactions themethod of the present invention captures an exponential mathematicaladvantage without producing additional copies of the target analyte. Ina simple invasive cleavage reaction the yield, Y, is simply the turnoverrate, K, multiplied by the time of the reaction, t (i.e., Y=(K)(t)). IfY is used to represent the yield of a simple reaction, then the yield ofa compound (i.e., a multiple, sequential reaction), assuming that eachof the individual invasive cleavage steps has the same turnover rate,can be simply represented as Y^(n), where n is the number of invasivecleavage reactions that have been performed in the series. If the yieldsof each step differ the ultimate yield can be represented as the productof the multiplication of the yields of each individual reaction in theseries. For example, if a primary invasive cleavage reaction can produceone thousand products in 30 minutes, and each of those products can inturn participate in 1000 additional reactions, there will be 1000²copies (1000×1000) of the ultimate product in a second reaction. If athird reaction is added to the series, then the theoretical yield willbe 1000³ (1000×1000×1000). In the methods of the present invention theexponent comes from the number of invasive cleavage reactions in thecascade. This can be contrasted to the amplification methods describedabove (e.g., PCR) in which Y is limited to 2 by the number of strands induplex DNA, and the exponent n is the number of cycles performed, sothat many iterations are necessary to accumulate large amounts ofproduct.

To distinguish the exponential amplifications described above from thoseof the present invention, the former can be considered reciprocatingreactions because the products the reaction feed back into the samereaction (e.g., event one leads to some number of events 2, and eachevent 2 leads back to some number of events 1). In contrast, the eventsof the present invention are sequential (e.g., event 1 leads to somenumber of events 2; each event 2 leads to some number of events 3, etc.,and no event can contribute to an event earlier in the chain).

The sensitivity of the reciprocating methods is also one of the greatestweaknesses when these assays are used to determine if a target nucleicacid sequence is present or absent in a sample. Because the product ofthese reactions is detectable copies of the starting material,contamination of a new reaction with the products of an earlier reactioncan lead to false positive results, (i.e., the apparent detection of thetarget nucleic acid in samples that do not actually contain any of thattarget analyte). Furthermore, because the concentration of the productin each positive reaction is so high, amounts of DNA sufficient tocreate a strong false positive signal can be communicated to newreactions very easily either by contact with contaminated instruments orby aerosol. In contrast to the reciprocating methods, the mostconcentrated product of the sequential reaction (i.e., the productreleased in the ultimate invasive cleavage event) is not capable ofinitiating a like reaction or cascade if carried over to a fresh testsample. This is a marked advantage over the exponential amplificationmethods described above because the reactions of the present inventionmay be performed without the costly containment arrangements (e.g.,either by specialized instruments or by separate laboratory space)required by any reciprocating reaction. While the products of apenultimate event may be inadvertently transferred to produce abackground of the ultimate product in the absence of the a targetanalyte, the contamination would need to be of much greater volume togive an equivalent risk of a false positive result.

When the term sequential is used it is not intended to limit theinvention to configurations in which that one invasive cleavage reactionor assay must be completed before the initiation of a subsequentreaction for invasive cleavage of a different probe. Rather, the termrefers to the order of events as would occur if only single copies ofeach of the oligonucleotide species were used in an assay. The primaryinvasive cleavage reaction refers to that which occurs first, inresponse to the formation of the cleavage structure on the targetnucleic acid. Subsequent reactions may be referred to as secondary,tertiary and so forth, and may involve artificial “target” strands thatserve only to support assembly of a cleavage structure, and which areunrelated to the nucleic acid analyte of interest. While the completeassay may, if desired, be configured with each step of invasive cleavageseparated either in space (e.g., in different reaction vessels) or intime (e.g., using a shift in reaction conditions, such as temperature,enzyme identity or solution condition, to enable the later cleavageevents), it is also contemplated that all of the reaction components maybe mixed so that secondary reactions may be initiated as soon as productfrom a primary cleavage becomes available. In such a format, primary,secondary and subsequent cleavage events involving different copies ofthe cleavage structures may take place simultaneously.

Several levels of this sort of linear amplification can be envisioned,in which each successive round of cleavage produces an oligonucleotidethat can participate in the cleavage of a different probe in subsequentrounds. The primary reaction would be specific for the analyte ofinterest with secondary (and tertiary, etc.) reactions being used togenerate signal while still being dependent on the primary reaction forinitiation.

The released product may perform in several capacities in the subsequentreactions. One of the possible variations is shown in FIG. 96, in whichthe product of one invasive cleavage reaction becomes the INVADERoligonucleotide to direct the specific cleavage of another probe in asecond reaction. In FIG. 96, the first invasive cleavage structure isformed by the annealing of the INVADER oligonucleotide (“Invader”) andthe probe oligonucleotide (“Probe 1”) to the first target nucleic acid(“Target 1”). The target nucleic acid is divided into three regionsbased upon which portions of the INVADER and probe oligonucleotides arecapable of hybridizing to the target (as discussed above and as shown inFIG. 25). Region 1 (region Y in FIG. 25) of the target hascomplementarity to only the INVADER oligonucleotide; region 3 (region Zin FIG. 25) of the target has complementarity to only the probe; andregion 2 (region X in FIG. 25) of the target has complementarity to boththe INVADER and probe oligonucleotides. It is noted that the sequentialinvasive cleavage reaction diagrammed in FIG. 96 employs an INVADER anda probe oligonucleotide; the sequential cleavage reaction is not limitedto the use of such a first cleavage structure. The first cleavagestructure in the sequential reaction may also employ an INVADERoligonucleotide, a mini probe and a stacker oligonucleotide as discussedabove. Further, as discussed above, the overlap in any or all of thecleavage structures in the sequential reactions may comprise moietiesother than overlapping complementary bases, such that the region shownas “X” represents a region where there is a physical rather thansequence overlap between the INVADER and probe oligonucleotides In FIG.96, cleavage of Probe 1 releases the “Cut Probe 1” (indicated by thehatched line in both the cleaved and uncleaved Probe 1 in FIG. 96). Thereleased Probe 1 is then used as the INVADER oligonucleotide in secondcleavage. The second cleavage structure is formed by the annealing ofthe Cut Probe 1, a second probe oligonucleotide (“Probe 2”) and a secondtarget nucleic acid (“Target 2”) In some embodiments, Probe 2 and thesecond target nucleic acid are covalently connected, preferably at their3′ and 5′ ends, respectively, thus forming a hairpin stem and loop,termed herein a “cassette”. The loop may be nucleic acid, (e.g., astring of nucleotides, such as the four T residues depicted in severalFigures, including 113A) or a non-nucleic acid spacer or linker.Inclusion of an excess of the cassette molecule allows each Cut Probe 1to serve as an INVADER to direct the cleavage of multiple copies of thecassette.

Probe 2 may be labeled (e.g., as indicated by the star in FIG. 96) anddetection of cleavage of the second cleavage structure may beaccomplished by detecting the labeled cut Probe 2; the label may aradioisotope (e.g., ³²P, ³⁵S), a fluorophore (e.g., fluorescein), areactive group capable of detection by a secondary agent (e.g.,biotin/streptavidin), a positively charged adduct which permitsdetection by selective charge reversal (as discussed in Section IVabove), etc. Alternatively, the cut Probe 2 may used in a tailingreaction, or to complete or activate a protein binding site, or may bedetected or used by any of the means for detecting or using anoligonucleotide described herein.

Another possible configuration for performing a sequential invasivecleavage reaction is diagrammed in FIG. 97. In this embodiment, probeoligonucleotides that are cleaved in the primary reaction can bedesigned to fold back on themselves (i.e., they contain a region ofself-complementarity) to create a molecule that can serve as both theINVADER and target oligonucleotide (termed here an “IT” complex). The ITcomplex then enables cleavage of a different probe present in thesecondary reaction. Inclusion of an excess of the secondary probemolecule (“Probe 2”), allows each IT molecule to serve as the platformfor the generation of multiple copies of cleaved secondary probe. InFIG. 97, the regions of self-complementarity contained within the 5′portion of the INVADER oligonucleotide is indicated by the hatchedovals; the arrow between these two ovals indicates that these tworegions can self-pair (as shown in the “Cut Probe 1”). The targetnucleic acid is divided into three regions based upon which portions ofthe INVADER and probe oligonucleotides are capable of hybridizing to thetarget (as discussed above and it is noted that the target may bedivided into four regions if a stacker oligonucleotide is employed). Thesecond cleavage structure is formed by the annealing of the second probe(“Probe 2”) to the fragment of Probe 1 (“Cut Probe 1”) that was releasedby cleavage of the first cleavage structure. The Cut Probe 1 forms ahairpin or stem/loop structure near its 3′ terminus by virtue of theannealing of the regions of self-complementarity contained within CutProbe 1 (this self-annealed Cut Probe 1 forms the IT complex). The ITcomplex (Cut Probe 1) is divided into three regions. Region 1 of the ITcomplex has complementarity to the 3′ portion of Probe 2; region 2 hascomplementarity to both the 3′ end of Cut Probe 1 and to the 5′ portionof Probe 2 (analogous to the region of overlap “X” shown in FIG. 25);and region 3 contains the region of self-complementarity (i.e., region 3is complementary to the 3′ portion of the Cut Probe 1). Note that withregard to the IT complex (i.e., Cut Probe 1), region 1 is locatedupstream of region 2 and region 2 is located upstream of region 3. Asfor other embodiments of invasive cleavage, the region shown as “2” canrepresent a region where there is a physical, but not sequence, overlapbetween the INVADER portion of the Cut Probe 1 and the Probe 2oligonucleotide.

The cleavage products of the secondary invasive cleavage reaction (i.e.,Cut Probe 2) can either be detected, or can in turn be designed toconstitute yet another integrated INVADER-target complex to be used witha third probe molecule, again unrelated to the preceding targets.

The present invention is not limited to the configurations diagrammed inFIGS. 96 and 97. It is envisioned that the oligonucleotide product of aprimary cleavage reaction may fill the role of any of theoligonucleotides described herein (e.g., it may serve as a target strandwithout an attached INVADER oligonucleotide-like sequence, or it mayserve as a stacker oligonucleotide, as described above), to enhance theturnover rate seen in the secondary reaction by stabilizing the probehybridization through coaxial stacking.

Secondary cleavage reactions in some preferred embodiments of thepresent invention include the use of FRET cassettes such as thosedescribed in Examples 54 through 62. Such molecules provide both asecondary target and a FRET labeled cleavable sequence, allowinghomogeneous detection (i.e., without product separation or othermanipulation after the reaction) of the sequential invasive cleavagereaction. Other preferred embodiments use a secondary reaction system inwhich the FRET probe and synthetic target are provided as separateoligonucleotides.

In a preferred embodiment, each subsequent reaction is facilitated by(i.e., is dependent upon) the product of the previous cleavage, so thatthe presence of the ultimate product may serve as an indicator of thepresence of the target analyte. However, cleavage in the second reactionneed not be dependent upon the presence of the product of the primarycleavage reaction; the product of the primary cleavage reaction maymerely measurably enhance the rate of the second cleavage reaction.

In summary, the INVADER assay cascade (i.e., sequential invasivecleavage reactions) of the present invention is a combination of two ormore linear assays that allows the accumulation of the ultimate productat an exponential rate, but without significant risk of carryovercontamination. It is an important to note that background that does notarise from sequential cleavage, such as thermal breakage of thesecondary probe, generally increases linearly with time. In contrast,signal generation from a 2-step sequential reaction follows quadratickinetics. Thus, collection of data as a time course, either by takingtime points or through the use of an instrument that allows real-timedetection during the INVADER assay reaction incubations, provides theattractive capability of discriminating between the true signal and anybackground solely on the basis of quadratic versus linear increases insignal over time. For example, when viewed graphically, the real signalwill appear as a quadratic curve, while any accumulating background willbe linear, and thus easy to distinguish, even if the absolute level ofthe background signal (e.g., fluorescence in a FRET detection format) issubstantial.

The sequential invasive cleavage amplification of the present inventioncan be used as an intermediate boost to any of the detection methods(e.g., gel based analysis by either standard or by charge reversal),polymerase tailing, and incorporation into a protein binding region,described herein. When used is such combinations the increasedproduction of a specific cleavage product in the invasive cleavage assayreduces the burdens of sensitivity and specificity on the read-outsystems, thus facilitating their use.

In addition to enabling a variety of detection platforms, the cascadestrategy is suitable for multiplex analysis of individual analytes(i.e., individual target nucleic acids) in a single reaction. Themultiplex format can be categorized into two types. In one case, it isdesirable to know the identity (and amount) of each of the analytes thatcan be present in a clinical sample, or the identity of each of theanalytes as well as an internal control. To identify the presence ofmultiple individual analytes in a single sample, several distinctsecondary amplification systems may be included. Each probe cleaved inresponse to the presence of a particular target sequence (or internalcontrol) can be designed to trigger a different cascade coupled todifferent detectable moieties, such as different sequences to beextended by DNA polymerase or different dyes in an FRET format. Thecontribution of each specific target sequence to final product canthereby be tallied, allowing quantitative detection of different genesor alleles in a sample containing a mixture of genes or alleles.

In the second configuration, it is desirable to determine if any ofseveral analytes are present in a sample, but the exact identity of eachdoes not need to be known. For example, in blood banking it is desirableto know if any one of a host of infectious agents is present in a sampleof blood. Because the blood is discarded regardless of which agent ispresent, different signals on the probes would not be required in suchan application of the present invention, and may actually be undesirablefor reasons of confidentiality. In this case, the 5′ arms (i.e., the 5′portion which will be released upon cleavage) of the differentanalyte-specific probes would be identical and would therefore triggerthe same secondary signal cascade. A similar configuration would permitmultiple probes complementary to a single gene to be used to boost thesignal from that gene or to ensure inclusivity when there are numerousalleles of a gene to be detected.

In the primary INVADER reaction, there are two potential sources ofbackground. The first is from INVADER-independent cleavage of probeannealed to the target, to itself, or to one of the otheroligonucleotides present in the reaction. It can be seen byconsideration of FIGS. 96 and 97 that the probes of the primary cleavagereactions depicted are designed to have regions of complementarity tothe other oligonucleotides involved in the subsequent reactions, and, asdepicted in FIG. 97, to other regions of the same molecule. The use ofan enzyme that cannot efficiently cleave a structure that lacks a primer(e.g., that cannot cleave the structures diagrammed in FIG. 16A or 16 D)is preferred for this reason. As shown in FIGS. 99 and 100, the enzymePfu u FEN-1 gives no detectable cleavage in the absence of the upstreamoligonucleotide or even in the presence of an upstream oligonucleotidethat fails to invade the probe-target complex. This indicates that thePfu u FEN-1 endonuclease is a suitable enzyme for use in the methods ofthe present invention.

Other structure-specific nucleases may be suitable as a well. Asdiscussed in the first example, some 5′ nucleases can be used inconditions that significantly reduce this primer-independent cleavage.For example, it has been shown that when the 5′ nuclease of DNAPTaq isused to cleave hairpins the primer-independent cleavage is markedlyreduced by the inclusion of a monovalent salt in the reaction(Lyamichev, et al., [1993], supra).

Test For INVADER Oligonucleotide-Independent Cleavage

A simple test can be performed for any enzyme in combination with anyreaction buffer to gauge the amount of INVADERoligonucleotide-independent cleavage to be expected from thatcombination. A small hairpin-like test molecule that can be used with orwithout a primer hybridized to a 3′ arm, the S-60 molecule, is depictedin FIG. 30. The S-60 and the oligonucleotide P15 are a convenient set ofmolecules for testing the suitability of an enzyme for application inthe present invention and conditions for using these molecules aredescribed in Example 11. Other similar hairpins may be used. A cleavagestructure may be assembled from separate oligonucleotides as diagrammedin FIGS. 99 a-e. Reactions using these structures to examine theactivity of the Pfu FEN-1 enzyme in the presence or absence of anupstream overlapping oligonucleotide are described in Example 45 and theresults are displayed in FIG. 100. To test any particular combination ofenzyme and cleavage conditions, similar reactions can be assembled.Outside of the variables of reaction conditions to be tested for anyparticular enzyme (e.g., salt sensitivities, divalent cationrequirements) the test reactions should accommodate any knownlimitations of the test enzyme. For example, the test reactions shouldbe performed at a temperature that is within the operating temperaturerange of the candidate enzyme, if known.

It is not necessary that multiple lengths of overlap be demonstrated foreach candidate enzyme, but the activity of the enzyme in the absence ofan upstream oligonucleotide (sequence or physical overlap) (as shown inFIG. 99 a) and in the presence of an oligonucleotide that does notoverlap (FIG. 99 b) should be assessed. It is preferable that structureslacking an upstream oligonucleotide be cleaved at less than one half ofthe rate seen in the presence of an upstream overlappingoligonucleotide. It is more preferable that these structures be cleavedat less than about on tenth the rate of the invasive cleavage structure.It is most preferred that cleavage of these structures occur at lessthan one percent the rate of the invasive cleavage structure.

If the cleaved product is to serve as an upstream oligonucleotide in asubsequent cleavage reaction, as diagrammed in FIG. 96, the most rapidreaction will be achieved if the other components of the second cleavagestructure (i.e., Target 2 and Probe 2 in FIG. 96) are provided in excesscompared to the amount of first cleavage product, so that cleavage mayproceed immediately after the upstream oligonucleotide (i.e, Cut Probe 1in FIG. 96) is made available. To provide an abundance of the secondtarget strand or cassette (Target 2 in FIG. 96) one may use an isolatednatural nucleic acid, such as bacteriophage M13 DNA, or one may use asynthetic oligonucleotide. If a synthetic oligonucleotide is chosen asthe second target sequence, the sequence employed should be examined forregions of unintended self-complementarity (similar considerations applyto short isolated natural nucleic acids such as restriction enzymefragments or PCR products; natural nucleic acid targets whose 3′ end islocated ≧100 nucleotides downstream of the probe binding site on thetarget strand are generally long enough to obviate the designconsiderations discussed below). Specifically, it should be determinedthat the 3′ end of the synthetic oligonucleotide may not hybridize tothe target strand (i.e., intra-strand hybridization) upstream of theprobe, triggering unintended cleavage. Simple examination of thesequence of the synthetic oligonucleotide should reveal if the 3′ endhas sufficient complementarity to the region of the target upstream ofthe probe binding site to pose a problem (i.e, it would reveal whetherthe synthetic oligonucleotide can form a hairpin at its 3′ end whichcould act as an invading oligonucleotide to cause cleavage of the 2^(nd)probe in the absence of the hybridization of the intended INVADERoligonucleotide (i.e., the cleavage product from the first invasivecleavage reaction)). If 3 or more of the last 4 to 7 nucleotides (the 3′terminal region) of the synthetic target can basepair upstream of theprobe such that there is an overlap with the probe-target duplex, orsuch that the duplexes formed by the synthetic target strand with itsown 3′ terminal region and with the probe abut without a gap and the 3′terminal region has an additional 1 or 2 nucleotides unpaired at theextreme 3′ end of the synthetic target, then the sequence of thesynthetic target oligonucleotide should be modified. The sequence may bechanged to disrupt the interaction of the 3′ terminal region or toincrease the distance between the probe binding site and the regions towhich the 3′ terminus is binding. Alternatively, the 3′ end may bemodified to reduce its ability to direct cleavage (e.g., by adding a 3′phosphate during synthesis) (see Ex. 35, Table 3) or by adding severaladditional nucleotides that will not basepair in a self-complementarymanner (i.e., they will not participate in the formation of a hairpinstructure).

When the product of a first invasive cleavage reaction is designed toform a target that can fold on itself to direct cleavage of a secondprobe, the IT complex as diagrammed in FIG. 97, the design of thesequence used to form the stem/loop of the IT complex should beconsidered. To be factored into the design of such a probe are 1) thelength of the region of self-complementarity, 2) the type of overlap(i.e., what 3′ moiety) and, if an overlap in sequence is selected, thelength of the region of overlap (region “X” in FIG. 25) and 3) thestability of the hairpin or stem/loop structure as predicted by bothWatson-Crick base pairing and by the presence or absence of aparticularly stable loop sequence (e.g., a tetraloop [Tinoco et al.,supra], or a triloop [Hirao et al., supra]). It is desirable that thissequence have nucleotides that can base pair (intrastrand), so that thesecond round of invasive cleavage may occur, but that the structure notbe so strong that its presence will prevent the cleavage of the probe inthe primary reaction (i.e., Probe 1 in FIG. 96). As shown herein, thepresence of a secondary structure in the 5′ arm of a cleavage structurecleaved by a structure-specific nuclease may inhibit cleavage by somestructure-specific nucleases (Ex. 1).

The length of the region of self-complementarity within Probe 1determines the length of the region of the duplex upstream of Probe 2 inthe second cleavage structure (see FIG. 97). Different enzymes havedifferent length requirements for this duplex to effect invasivecleavage efficiently. For example, the Pfu u FEN-1 and Mja FEN-1 enzymeshave been tested for the effect of this duplex length using the set oftarget/INVADER oligonucleotide molecules depicted in FIG. 98 (i.e., SEQID NOS:118, 119, 147-151). The invasive cleavage reactions wereperformed as described in Example 38, using 1 pM IT3 (SEQ ID NO:118), 2μM probe PR1 (SEQ ID NO:119) for 5 min, and the rates of cleavage areshown in Table 2.

TABLE 2 Pfu FEN-1 Turnover, Mja FEN-1 Turnover, per Length of Duplex permin. min. 0 0 0 3 1 29 4 10 57 6 44 51 8 45 46

The data shown in Table 2 demonstrate that the Pfu FEN-1 enzyme can beused with stems of 3 or 4 bases, but that the rate of cleavage ismaximized when the stem is greater than 4 basepairs in length. Table 2shows that the Mja FEN-1 enzyme can cleave efficiently using shorterstems; however, as this enzyme can also cleave a probe in the absence ofan upstream oligonucleotide, Mja FEN-1 is not preferred for use in thesequential invasive cleavage methods of the present invention.

A similar test can be performed using any candidate enzyme to determinehow much self-complementarity may be designed into the Probe 1. The useof a shorter stem means that the overall probe may be shorter. This isbeneficial because shorter probes are less costly to synthesize, andbecause shorter probes will have fewer sequences that might formunintended intrastrand structures. In assessing the activity of acandidate enzyme on the structures such as those shown in FIG. 98 it isnot required that the stem length chosen allow the maximum rate ofcleavage to occur. For example, in considering the case of Pfu FEN-1,the advantages of using a 4 basepair stem (e.g., cost or sequencelimitations), with a cleavage rate of 10 cleavages per minute, mayoutweigh the rate advantage of using a longer 6 basepair stem (44cleavages/min.), in the context of a particular experiment. It is withinthe scope of the present invention that some elements chosen for use inthe assay be sub-optimal for performance of that particular element, ifthe use of a sub-optimal design benefits the objectives of thatparticular experiment as a whole.

In designing oligonucleotides to be employed as a probe that, oncecleaved, forms a stem-loop structure as diagrammed in FIG. 97 (i.e.,Probe 1 in FIG. 97), it has been found that the stability of the loop isnot a factor in the efficiency of cleavage of either Probe 1 or Probe 2.Loops tested have included stable triloops, loops of 3 and 4 nucleotidesthat were not predicted to be particularly stable (i.e., the stabilityis determined by the duplex sequence and not by additional stabilizinginteractions within the loop), and large loops of up to about 25nucleotides.

IV. Fractionation of Specific Nucleic Acids by Selective Charge Reversal

Some nucleic acid-based detection assays involve the elongation and/orshortening of oligonucleotide probes. For example, as described herein,the primer-directed, primer-independent, and INVADER-directed cleavageassays, as well as the “nibbling” assay all involve the cleavage (i.e.,shortening) of oligonucleotides as a means for detecting the presence ofa target nucleic sequence. Examples of other detection assays thatinvolve the shortening of an oligonucleotide probe include the “TaqMan”or nick-translation PCR assay described in U.S. Pat. No. 5,210,015 toGelfand et al. (the disclosure of which is herein incorporated byreference), the assays described in U.S. Pat. Nos. 4,775,619 and5,118,605 to Urdea (the disclosures of which are herein incorporated byreference), the catalytic hybridization amplification assay described inU.S. Pat. No. 5,403,711 to Walder and Walder (the disclosure of which isherein incorporated by reference), and the cycling probe assay describedin U.S. Pat. Nos. 4,876,187 and 5,011,769 to Duck et al. (thedisclosures of which are herein incorporated by reference). Examples ofdetection assays that involve the elongation of an oligonucleotide probe(or primer) include the polymerase chain reaction (PCR) described inU.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et al. (thedisclosures of which are herein incorporated by reference) and theligase chain reaction (LCR) described in U.S. Pat. Nos. 5,427,930 and5,494,810 to Birkenmeyer et al. and Barany et al. (the disclosures ofwhich are herein incorporated by reference). The above examples areintended to be illustrative of nucleic acid-based detection assays thatinvolve the elongation and/or shortening of oligonucleotide probes anddo not provide an exhaustive list.

Typically, nucleic acid-based detection assays that involve theelongation and/or shortening of oligonucleotide probes requirepost-reaction analysis to detect the products of the reaction. It iscommon that the specific reaction product(s) must be separated from theother reaction components, including the input or unreactedoligonucleotide probe. One detection technique involves theelectrophoretic separation of the reacted and unreacted oligonucleotideprobe. When the assay involves the cleavage or shortening of the probe,the unreacted product will be longer than the reacted or cleavedproduct. When the assay involves the elongation of the probe (orprimer), the reaction products will be greater in length than the input.Gel-based electrophoresis of a sample containing nucleic acid moleculesof different lengths separates these fragments primarily on the basis ofsize. This is due to the fact that in solutions having a neutral oralkaline pH, nucleic acids having widely different sizes (i.e.,molecular weights) possess very similar charge-to-mass ratios and do notseparate (Andrews, Electrophoresis, 2nd Edition, Oxford University Press(1986), pp. 153-154]. The gel matrix acts as a molecular sieve andallows nucleic acids to be separated on the basis of size and shape(e.g., linear, relaxed circular or covalently closed supercoiledcircles).

Unmodified nucleic acids have a net negative charge due to the presenceof negatively charged phosphate groups contained within thesugar-phosphate backbone of the nucleic acid. Typically, the sample isapplied to gel near the negative pole and the nucleic acid fragmentsmigrate into the gel toward the positive pole with the smallestfragments moving fastest through the gel.

The present invention provides a novel means for fractionating nucleicacid fragments on the basis of charge. This novel separation techniqueis related to the observation that positively charged adducts can affectthe electrophoretic behavior of small oligonucleotides because thecharge of the adduct is significant relative to charge of the wholecomplex. In addition to the use of positively charged adducts (e.g., Cy3and Cy5 fluorescent dyes, the positively charged heterodimericDNA-binding dyes shown in FIG. 66, etc.), the oligonucleotide maycontain amino acids (particularly useful amino acids are the chargedamino acids: lysine, arginine, asparate, glutamate), modified bases,such as amino-modified bases, and/or a phosphonate backbone (at all or asubset of the positions). In other embodiments, as discussed furtherbelow, a neutral dye or detection moiety (e.g., biotin, streptavidin,etc.) may be employed in place of a positively charged adduct, inconjunction with the use of amino-modified bases and/or a complete orpartial phosphonate backbone.

This observed effect is of particular utility in assays based on thecleavage of DNA molecules. Using the assays described herein as anexample, when an oligonucleotide is shortened through the action of aCLEAVASE enzyme or other cleavage agent, the positive charge can be madeto not only significantly reduce the net negative charge, but toactually override it, effectively “flipping” the net charge of thelabeled entity. This reversal of charge allows the products oftarget-specific cleavage to be partitioned from uncleaved probe byextremely simple means. For example, the products of cleavage can bemade to migrate towards a negative electrode placed at any point in areaction vessel, for focused detection without gel-basedelectrophoresis; Example 24 provides examples of devices suitable forfocused detection without gel-based electrophoresis. When a slab gel isused, sample wells can be positioned in the center of the gel, so thatthe cleaved and uncleaved probes can be observed to migrate in oppositedirections. Alternatively, a traditional vertical gel can be used, butwith the electrodes reversed relative to usual DNA gels (i.e., thepositive electrode at the top and the negative electrode at the bottom)so that the cleaved molecules enter the gel, while the uncleaveddisperse into the upper reservoir of electrophoresis buffer.

An important benefit of this type of readout is the absolute nature ofthe partition of products from substrates (i.e., the separation isvirtually 100%). This means that an abundance of uncleaved probe can besupplied to drive the hybridization step of the probe-based assay, yetthe unconsumed (i.e., unreacted) probe can, in essence, be subtractedfrom the result to reduce background by virtue of the fact that theunreacted probe will not migrate to the same pole as the specificreaction product.

Through the use of multiple positively charged adducts, syntheticmolecules can be constructed with sufficient modification that thenormally negatively charged strand is made nearly neutral. When soconstructed, the presence or absence of a single phosphate group canmean the difference between a net negative or a net positive charge.This observation has particular utility when one objective is todiscriminate between enzymatically generated fragments of DNA, whichlack a 3′ phosphate, and the products of thermal degradation, whichgenerally retain a 3′ phosphate (and thus two additional negativecharges). Examples 22 and 23 demonstrate the ability to separatepositively charged reaction products from a net negatively chargedsubstrate oligonucleotide. As discussed in these examples,oligonucleotides may be transformed from net negative to net positivelycharged compounds. In Example 23, the positively charged dye, Cy3 wasincorporated at the 5′ end of a 22-mer (SEQ ID NO:50) which alsocontained two amino-substituted residues at the 5′ end of theoligonucleotide; this oligonucleotide probe carries a net negativecharge. After cleavage, which occurred 2 nucleotides into the probe, thefollowing labeled oligonucleotide was released: 5′-Cy3-AminoT-AminoT-3′(in addition to unlabeled fragment comprising the remaining 20nucleotides of SEQ ID NO:50). This short fragment bears a net positivecharge while the remainder of the cleaved oligonucleotide and theunreacted or input oligonucleotide bear net negative charges.

The present invention contemplates embodiments wherein the specificreaction product produced by any cleavage of any oligonucleotide can bedesigned to carry a net positive charge while the unreacted probe ischarge neutral or carries a net negative charge. The present inventionalso contemplates embodiments where the released product may be designedto carry a net negative charge while the input nucleic acid carries anet positive charge. Depending on the length of the released product tobe detected, positively charged dyes may be incorporated at the one endof the probe and modified bases may be placed along the oligonucleotidesuch that upon cleavage, the released fragment containing the positivelycharged dye carries a net positive charge. Amino-modified bases may beused to balance the charge of the released fragment in cases where thepresence of the positively charged adduct (e.g., dye) alone is notsufficient to impart a net positive charge on the released fragment. Inaddition, the phosphate backbone may be replaced with a phosphonatebackbone at a level sufficient to impart a net positive charge (this isparticularly useful when the sequence of the oligonucleotide is notamenable to the use of amino-substituted bases); FIGS. 45 and 46 showthe structure of short oligonucleotides containing a phosphonate groupon the second T residue). An oligonucleotide containing a fullyphosphonate-substituted backbone would be charge neutral (absent thepresence of modified charged residues bearing a charge or the presenceof a charged adduct) due to the absence of the negatively chargedphosphate groups. Phosphonate-containing nucleotides (e.g.,methylphosphonate-containing nucleotides are readily available and canbe incorporated at any position of an oligonucleotide during synthesisusing techniques which are well known in the art.

In essence, the invention contemplates the use of charge-basedseparation to permit the separation of specific reaction products fromthe input oligonucleotides in nucleic acid-based detection assays. Thefoundation of this novel separation technique is the design and use ofoligonucleotide probes (typically termed “primers” in the case of PCR)which are “charge balanced” so that upon either cleavage or elongationof the probe it becomes “charge unbalanced,” and the specific reactionproducts may be separated from the input reactants on the basis of thenet charge.

In the context of assays that involve the elongation of anoligonucleotide probe (i.e., a primer), such as is the case in PCR, theinput primers are designed to carry a net positive charge. Elongation ofthe short oligonucleotide primer during polymerization will generate PCRproducts that now carry a net negative charge. The specific reactionproducts may then easily be separated and concentrated away from theinput primers using the charge-based separation technique describedherein (the electrodes will be reversed relative to the description inExample 23 as the product to be separated and concentrated after a PCRwill carry a negative charge).

V. Signal Enhancement by Tailing of Reaction Products in the INVADEROligonucleotide-Directed Cleavage Assay

It has been determined that when oligonucleotide probes are used incleavage detection assays at elevated temperature, some fraction of thetruncated probes will have been shortened by nonspecific thermaldegradation, and that such breakage products can make the analysis ofthe target-specific cleavage data more difficult. The thermaldegradation that creates a background ladder of bands when the probes ofthe present invention are treated at high temperature for more than afew minutes occurs as a two step process. In the first step theN-glycosyl bond breaks, leaving an abasic site in the DNA strand. At theabasic site the DNA chain is weakened and undergoes spontaneous cleavagethrough a beta-elimination process. It has been determined that purinebases are about 20 times more prone to breakage than pyrimidine bases(Lindahl, Nature 362:709 [1993]). This suggests that one way of reducingbackground in methods using oligonucleotides at elevated temperatures isto select target sequences that allow the use of pyrimidine-rich probes.It is preferable, where possible, to use oligonucleotides that areentirely composed of pyrimidine residues. If only one or a few purinesare used, the background breakage will appear primarily at thecorresponding sites, and these bands (due to thermal breakdown) may bemistaken for the intended cleavage products if care is not taken in thedata analysis (i.e., proper controls must be run).

Background cleavage due to thermal breakdown of probe oligonucleotidescan, when not resolved from specific cleavage products, reduce theaccuracy of quantitation of target nucleic acids based on the amount ofaccumulated product in a set timeframe. One means of distinguishing thespecific from the nonspecific products is disclosed above, and is basedon partitioning the products of these reactions by differences in thenet charges carried by the different molecular species in the reaction.As was noted in that discussion, the thermal breakage products usuallyretain 3′ phosphates after breakage, while the enzyme-cleaved productsdo not. The two negative charges on the phosphate facilitatecharge-based partition of the products.

The absence of a 3′ phosphate on the desired subset of the probefragments may be used to advantage in enzymatic assays as well. Nucleicacid polymerases, both non-templated (e.g., terminal deoxynucleotidyltransferase, polyA polymerase) and template-dependent (e.g., Pol I-typeDNA polymerases), require an available 3′ hydroxyl by which to attachfurther nucleotides. This enzymatic selection of 3′ end structure may beused as an effective means of partitioning specific from non-specificproducts.

In addition to the benefits of the partitioning described above, theaddition of nucleotides to the end of the specific product of an INVADERoligonucleotide-specific cleavage offers an opportunity to either addlabel to the products, to add capturable tails to facilitatesolid-support based readout systems, or to do both of these things atthe same time. Some possible embodiments of this concept are illustratedin FIG. 56.

In FIG. 56, an INVADER cleavage structure comprising an INVADERoligonucleotide containing a blocked or non-extendible 3′ end (e.g., a3′ dideoxynucleotide) and a probe oligonucleotide containing a blockedor non-extendable 3′ end (the open circle at the 3′ end of theoligonucleotides represents a non-extendible nucleotide) and a targetnucleic acid is shown; the probe oligonucleotide may contain a 5′ endlabel such as a biotin or a fluorescein (indicated by the stars) label(cleavage structures which employ a 5′ biotin-labeled probe or a 5′fluorescein-labeled probe are shown below the large diagram of thecleavage structure to the left and the right, respectively). Followingcleavage of the probe (the site of cleavage is indicated by the largearrowhead), the cleaved biotin-labeled probe is extended using atemplate-independent polymerase (e.g., TdT) and fluoresceinatednucleotide triphosphates. The fluorescein tailed cleaved probe moleculeis then captured by binding via its 5′ biotin label to streptavidin andthe fluorescence is then measured. Alternatively, following, cleavage ofa 5′-fluoresceinated probe, the cleaved probe is extended using atemplate-independent polymerase (e.g., TdT) and dATP. The polyadenylated(A-tailed) cleaved probe molecule is then captured by binding via thepolyA tail to oligo dT attached to a solid support.

The examples described in FIG. 56 are based on the use of TdT to tailthe specific products of INVADER-directed cleavage. The description ofthe use of this particular enzyme is presented by way of example and isnot intended as a limitation (indeed, when probe oligonucleotidescomprising RNA are employed, cleaved RNA probes may be extended usingpolyA polymerase). It is contemplated that an assay of this type can beconfigured to use a template-dependent polymerase, as described above.While this would require the presence of a suitable copy templatedistinct from the target nucleic acid, on which the truncatedoligonucleotide could prime synthesis, it can be envisaged that a probethat before cleavage would be unextendible, due to either mismatch ormodification of the 3′ end, could be activated as a primer when cleavedby an INVADER oligonucleotide-directed cleavage. A template directedtailing reaction also has the advantage of allowing greater selectionand control of the nucleotides incorporated.

The use of nontemplated tailing does not require the presence of anyadditional nucleic acids in the detection reaction, avoiding one step ofassay development and troubleshooting. In addition, the use ofnon-templated synthesis eliminated the step of hybridization,potentially speeding up the assay. Furthermore, the TdT enzyme is fast,able to add at least >700 nucleotides to substrate oligonucleotides in a15 minute reaction.

As mentioned above, the tails added can be used in a number of ways. Itcan be used as a straight-forward way of adding labeled moieties to thecleavage product to increase signal from each cleavage event. Such areaction is depicted in the left side of FIG. 66. The labeled moietiesmay be anything that can, when attached to a nucleotide, be added by thetailing enzyme, such as dye molecules, haptens such as digoxigenin, orother binding groups such as biotin.

In a preferred embodiment the assay includes a means of specificallycapturing or partitioning the tailed INVADER oligonucleotide-directedcleavage products in the mixture. It can be seen that target nucleicacids in the mixture may be tailed during the reaction. If a label isadded, it is desirable to partition the tailed INVADERoligonucleotide-directed cleavage products from these other labeledmolecules to avoid background in the results. This is easily done ifonly the cleavage product is capable of being captured. For example,consider a cleavage assay of the present invention in which the probeused has a biotin on the 5′ end and is blocked from extension on the 3′end, and in which a dye is added during tailing. Consider further thatthe products are to be captured onto a support via the biotin moiety,and the captured dye measured to assess the presence of the targetnucleic acid. When the label is added by tailing, only the specificallycleaved probes will be labeled. The residual uncut probes can still bindin the final capture step, but they will not contribute to the signal.In the same reaction, nicks and cuts in the target nucleic acid may betailed by the enzyme, and thus become dye labeled. In the final capturethese labeled targets will not bind to the support and thus, althoughlabeled, they will not contribute to the signal. If the final specificproduct is considered to consist of two portions, the probe-derivedportion and the tail portion, it can be seen from this discussion thatit is particularly preferred that, when the probe-derived portion isused for specific capture, whether by hybridization,biotin/streptavidin, or other method, that the label be associated withthe tail portion. Conversely, if a label is attached to theprobe-derived portion, then the tail portion may be made suitable forcapture, as depicted on the right side of FIG. 66. Tails may be capturedin a number of ways, including hybridization, biotin incorporation withstreptavidin capture, or by virtue if the fact that the longer moleculesbind more predictably and efficiently to a number of nucleic acidminding matrices, such as nitrocellulose, nylon, or glass, in membrane,paper, resin, or other form. While not required for this assay, thisseparation of functions allows effective exclusion from signal of bothunreacted probe and tailed target nucleic acid.

In addition to the supports described above, the tailed products may becaptured onto any support that contains a suitable capture moiety. Forexample, biotinylated products are generally captured withavidin-treated surfaces. These avidin surfaces may be in microtitreplate wells, on beads, on dipsticks, to name just a few of thepossibilities. Such surfaces can also be modified to contain specificoligonucleotides, allowing capture of product by hybridization. Capturesurfaces as described herein are generally known to those skilled in theart and include nitrocellulose dipsticks (e.g., GENECOMB, BioRad,Hercules, Calif.).

VI. Signal Enhancement by Completion of an Activated Protein BindingSite

In addition to the DNA polymerase tailing reaction described above, thepresent invention also contemplates the use of the products of theinvasive cleavage reaction to form activated protein binding sites, suchas RNA polymerase promoter duplexes, thereby allowing the interaction ofthe completed site to be used as an indicator of the presence of thenucleic acid that is the target of the invasive cleavage reaction. Byway of example, when an RNA polymerase promoter duplex is activated bybeing made complete (i.e., double-stranded over that portion of thepromoter region required for polymerase binding) through thehybridization of the oligonucleotide product of the invasive cleavagereaction, the synthesis of RNA can be used as such an indicator.

It is not intended that the transcription reaction of the presentinvention be limited to the use of any particular RNA polymerase or RNApolymerase promoter region. Promoter sequences are well characterizedfor several bacteriophage, including bacteriophage SP6, T7 and T3. Inaddition, promoter sequences have been well characterized for a numberof both eukaryotic and prokaryotic RNA polymerases. In a preferredembodiment, the promoter used enables transcription from one of thebacteriophage RNA polymerases. In a particularly preferred embodiment,the promoter used enables transcription by T7 RNA polymerase. Means ofperforming transcription in vitro are well known in the art andcommercial kits are available for performing transcription witheukaryotic, prokaryotic or bacteriophage RNA polymerases (e.g., fromPromega Corp., Madison, Wis.).

The protein binding regions of the present invention are not limited tothe bacteriophage RNA polymerase promoters described above. Otherpromoter sequences that are contemplated are those of prokaryotes andeukaryotes. For example, many strains of bacteria and fungi are used forthe expression of heterologous proteins. The minimal promoters requiredfor transcription by the RNA polymerases of organisms such as yeast andother fungi, eubacteria, nematodes, and cultured mammalian cells arewell described in the literature and in the catalogs of commercialsuppliers of DNA vectors for the expression of foreign proteins in theseorganisms.

The binding sites for other types of nucleic acid (e.g., DNA) bindingproteins are contemplated for use in the present invention. For example,proteins involved in the regulation of genes exert their effects bybinding to the DNA in the vicinity of the promoter from which the RNAfrom that gene is transcribed. The lac operator of E. coli is oneexample of a particularly well characterized and commonly used generegulation system in which the lac repressor protein binds to specificsequences that overlap, and thus block, the promoter for the genes underthe repressor's control (Jacob and Monod, Cold Spring Harbor Symposiumon Quantitative Biol. XXVI:193-211 [1961]). Many similar systems havebeen described in bacteria, including the trp and AraC regulatorysystems. Given the large amount of information available about bacterialpromoters, the steps described below for the design of suitable partialpromoters for the bacteriophage RNA polymerases can be readily adaptedto the design of detection systems based on these other promoters.

As noted above, many of the bacterial promoters are under the control ofa repressor or other regulatory protein. It is considered to be withinthe scope of the present invention to include the creation of compositebinding sites for these regulatory proteins through the provision of anucleic acid fragment (e.g., a non-target cleavage product generated inan invasive cleavage reaction). The binding of the regulatory protein tothe completed protein binding region (e.g., the composite bindingregion) can be assessed by any one of a number of means, includingslowed electrophoretic migration of either the protein or the DNAfragment, or by a conformational change in the protein or DNA uponbinding. In addition, transcription from a downstream promoter can bemonitored for up- or down-regulation as a result of the binding of theregulatory protein to the completed protein binding region.

In addition to the bacterial systems described above, many genes ineukaryotic systems have also been found to be under the control ofspecific proteins that bind to specific regions of duplex DNA. Examplesinclude, but are not limited to, the OCT-1, OCT-2 and AP-4 proteins inmammals and the GAL4 and GCN4 proteins in yeast. Such regulatoryproteins usually have a structural motif associated with duplex nucleicacid binding, such as a helix-turn-helix, a zinc finger or a leucinezipper [for review, see, Molecular and Cellular Biology, Wolfe (Ed.),Wadsworth Publishing Co., Belmont, Calif., pp. 694-715 [1993]).

For simplicity the test reaction described here will refer to T7 RNApolymerase, and its promoter. This is not intended to limit theinvention to the use of this RNA polymerase, and those skilled in theart of molecular biology would be able to readily adapt this describedtest to the examination of any of the DNA binding proteins, RNApolymerases and their binding or promoter sites discussed above.

It is known in the art that active T7 promoters can be formed by thehybridization of two oligonucleotides, each comprising either the top orbottom strand of the promoter sequence, such that a complete un-nickedduplex promoter is formed (Milligan et al., Nucl. Acids Res., 15:21,8783-8798 (1987)]. The present invention shows that one way of makingthe initiation of transcription dependent on the products of an invasivecleavage reaction is to design the probe for the cleavage reaction suchthat a portion of an RNA polymerase promoter is released as product. Theremaining DNA piece or pieces required to assemble a promoter duplex mayeither be provided as elements in the reaction mixture, or they may beproduced by other invasive cleavage events. If the oligonucleotidepieces are designed to comprise appropriate regions of complementaritythey may base pair to form a complete promoter duplex composed of threeor more nucleic acid fragments, as depicted in FIG. 88B. A promoterassembled in this way will have nicks in the backbone of one or bothstrands. In one embodiment, these nicks may be covalently closed throughthe use of a DNA ligase enzyme. In a preferred embodiment, the nicks arepositioned such that transcription can proceed without ligation. Inselecting the site of a nick created by the assembly of the partialpromoter fragment, at least one nick should be within the recognizedpromoter region for the RNA polymerase to be used. When a bacteriophagepromoter is used, a nick should be between nucleotides −17 and −1,measured from the site of transcription initiation at +1. In a preferredembodiment, a nick will be between nucleotides −13 and −8. In aparticularly preferred embodiment, a nick will be between nucleotides−12 and −10 on the non-template strand of the bacteriophage promoter.

When nicks are to be left unrepaired (i.e., not covalently closed with aDNA ligase) it is important to assess the effect of the nick location onthe level of transcription from the assembled promoter. A simple test isto combine the oligonucleotides that comprise the separate portions ofthe promoter with an oligonucleotide that comprises one entire strand ofthe promoter to be assembled, thereby forming a duplex promoter with anick in one strand. If the nick is in the top, or non-template strand ofthe promoter, then the oligonucleotide that comprises the completepromoter is made to include additional non-promoter sequence on its 5′end to serve as a template to be copied in the transcription. Thisarrangement is depicted in FIG. 88B. Alternatively, if the nick is to bein the bottom, or template strand of the promoter, then the partialpromoter oligonucleotide that covers the +1 position, the transcriptionstart site, will include the additional template sequence. Thisarrangement is depicted in FIGS. 95A-D (this Figure shows severaldifferent embodiments in which a cut probe or non-target cleavageproduct is used to form a composite promoter which contains one or morenicks on the template strand). In either case, the separateoligonucleotides are combined to form the complete promoter, and theassembly is used in a transcription reaction to create RNA.

To measure the effect of the nick, a substantially identical promoterfragment is created by hybridization of two oligonucleotides that eachcomprise one strand of the full-length promoter to create an un-nickedversion of the same promoter. These two molecular assemblies are testedin parallel transcription reactions and the amount of the expected RNAthat is produced in each reaction is measured for both size and yield. Apreferred method of assessing the size of the RNA is by electrophoresiswith subsequent visualization. If a labeled nucleotide (e.g., ³²P-GTP,or fluorescein-UTP) is used in the transcription, the RNA can bedetected and quantitated by autoradiography, fluorescence imaging or bytransfer to support membrane with subsequent detection (e.g., byantibody or hybridization probing). Alternatively, if unlabeled RNA isproduced the amounts may be determined by other methods known in theart, such as by spectrophotometry or by electrophoresis with subsequentstaining and comparison to known standards.

If the size of the RNA is as predicted by the template sequence, or ifit matches that produced from the control promoter, it can be presumedto have initiated transcription at the same site in the complex, and tohave produced essentially the same RNA product. If the product is muchshorter then transcription is either initiating at an internal site oris terminating prematurely (Schenborn and Mierendorf, Nucl. Acids Res.,13:17, 6223 [1985]; and Milligan et al., supra.). While this does notindicate that the assembly tested is completely unsuitable for theassay, the partial transcripts will reduce the gross amount of RNAcreated, perhaps compromising the signal from the assay, and suchproducts would require further characterization (e.g., finger printingor sequencing) to identify the nucleotide content of the product. It ispreferred that the size of the RNA produced matches that of the RNAproduced in the control reaction.

The yield of the reaction is also examined. It is not necessary that thelevel of transcription matches that of the control reaction. In someinstances (see Ex. 41, below) the nicked promoter may have an enhancedrate of transcription, while in other arrangements transcription may bereduced (relative to the rate from the un-nicked promoter assembly). Itis only required that the amount of product be within the detectionlimits of the method to be used with the test promoter.

It is reported that transcription from a bacteriophage promoter canproduce 200 to 1000 copies of each transcription template (template plusactive promoter) in a reaction. These levels of transcription are notrequired by the present invention. Reactions in which one RNA isproduced for each template are also contemplated.

The test described above will allow a promoter with a nick in anyposition to be assessed for utility in this assay. It is an objective ofthis invention to provide one or more of the oligonucleotides thatcomprise a partial promoter region through invasive cleavage event(s).In this embodiment, the partial promoter sequences are attached to theprobe oligonucleotide in the invasive cleavage assay, and are releasedby cleavage at specific site, as directed by the INVADERoligonucleotide. It is also intended that transcription be very poor ornonexistent in the absence of the correctly cleaved probe. To assess thesuccess of any oligonucleotide design at meeting these objectives,several transcription reaction tests can be performed.

For a promoter assembly that will have a nick on the non-templatestrand, several partial assemblies that should be tested are shown inFIGS. 86 A-D. By way of example, but not by way of limitation, thisFigure depicts the tests for a nicked promoter in which the upstream, or5′ portion of the non-template strand is to be provided by the invasivecleavage assay. This fragment is seen in FIG. 86A labeled as “cutprobe”. Transcription reactions incubated in the presence of the duplexshown in FIG. 86A will test the ability of the upstream partial promoterto allow initiation of transcription when hybridized to a bottom strand,termed a “copy template.” Similarly, a reaction performed in thepresence of the duplex depicted in FIG. 86B will test the ability of thepartial promoter fragment nearest the initiation site (the +1 site, asindicated in FIG. 85B) to support transcription of the copy template. Itis an important feature of the present invention that neither of thesepartial promoter duplexes be able to support transcription at the samelevel as would by seen in transcription from an intact promoter asdepicted in FIG. 85B. It is preferred that neither of these partialpromoters be sufficient to initiate detectable transcription in the timecourse of an average transcription reaction (i.e., within about an hourof incubation).

FIGS. 86C and 86D depict two other duplex arrangements designed to testthe effect of uncut probe within the transcription reaction. FIG. 86Cdepicts the duplex formed between only the uncut probe and the copytemplate, while FIG. 86D includes the other portion of the promoter. The3′ region of the probe is not complementary to the promoter sequence andtherefore produces an unpaired branch in the middle of the promoter. Itis an important feature of the present invention that neither of thesebranched promoter duplexes be able to support transcription at the samelevel as would by seen in transcription from an intact promoter asdepicted in FIG. 85B. It is preferred that neither of these branchedpromoters be sufficient to initiate detectable transcription in the timecourse of an average transcription reaction (i.e., within about an hourof incubation).

In one embodiment of the transcription system of the present invention,the initiation of transcription from the copy template in the absence ofa complete promoter, or in the presence of a branched promoter, isprevented by the judicious placement of the nick or nicks in thecomposite promoter. For example, as shown in the examples below,placement of a nick between the −12 and −11 nucleotides of thenon-template strand of the bacteriophage T7 promoter allowstranscription to take place only when the probe has been successfullycut, as in an invasive cleavage reaction. However, in some instanceswhere the invasive cleavage reaction is to provide the upstream portionof the non-template strand of the promoter (e.g., as depicted in FIG.88B) it may be necessary or desirable to place the nick on that strandin a particular position for reasons other than providing an optimalcomposite promoter (i.e., one that is inactive in the absence of any oneof the promoter pieces). It may be necessary or desirable to place thenick in such a way that the creation of a branched complete promoter(FIG. 86D) has an undesirable level of transcription, reducingdependence of RNA production on the success of the invasive cleavagestep. It is shown in the examples below that transcription from such abranched promoter can be suppressed by a modification of the downstreamnon-template promoter piece, shown as the “Partial PromoterOligonucleotide” in FIGS. 86, 88, 90 and 95D. As depicted in FIG. 90,the partial promoter oligonucleotide can be provided with a 5′ “tail” ofnucleotides that are not complementary to the template strand of thepromoter, but that are complementary to the 3′ portion of the probeoligonucleotide that would be removed in the invasive cleavage reaction.When uncut probe hybridizes to the copy template with the bound 5′tailed partial promoter oligonucleotide, the 5′ tail can basepair to the3′ region of the probe, forming a three-way junction as depicted in FIG.90A. This can effectively shut off transcription, as shown below. When acut probe hybridizes, as shown in FIG. 90B, a promoter with a smallbranch is formed, and it is shown herein that such a branched promotercan initiate transcription. Furthermore, if care is taken in selectingthe sequence of the 5′ tail (i.e., if the first unpaired base is thesame nucleotide at the 3′ nucleotide of the cut probe, so that theycompete for hybridization to the same template strand base), theresulting branched structure may also be cleaved by one of the structurespecific nucleases of the present invention, creating the un-branchedpromoter depicted in FIG. 90C, in some instances enhancing transcriptionover that seen with the FIG. 90B promoter.

The promoter duplex that is intended to be created, in this embodiment,by the successful execution of the INVADER directed cleavage assay willinclude both the “cut probe” and the partial promoter oligonucleotidedepicted in FIGS. 86A and B, aligned on a single copy template nucleicacid. The testing of the efficiency of transcription of such a nickedpromoter segment in comparison to the intact promoter is describedabove. All of the oligonucleotides described for these test moleculesmay be created using standard synthesis chemistries.

The set of test molecules depicted in FIG. 86 is designed to assess thetranscription capabilities of the variety of structures that may bepresent in reactions in which the 5′ portion of the non-template strandof the promoter is to be supplied by the INVADER directed cleavage. Itis also envisioned that a different portion of partial promoter may besupplied by the invasive cleavage reaction (e.g., the downstream segmentof the non-template strand of the promoter), as is shown in FIG. 94.Portions of the template strand of the promoter may also be provided bythe cut probe, as shown in FIGS. 95A-D. An analogous set of testmolecules, including “cut” and uncut versions of the probe to be used inthe invasive cleavage assay may be created to test any alternativedesign, whether the nick is to be located on the template or nontemplate strand of the promoter.

The transcription-based visualization methods of the present inventionmay also be used in a multiplex fashion. Reactions can be constructedsuch that the presence of one particular target leads to transcriptionfrom one type of promoter, while the presence of a different targetsequence (e.g., a mutant or variant) or another target suspected ofbeing present, may lead to transcription from a different (i.e., asecond) type of promoter. In such an embodiment, the identity of thepromoter from which transcription was initiated could be deduced fromthe type or size of the RNA produced.

By way of example, but not by way of limitation, the bacteriophagepromoters can be compared with such an application in view. Thepromoters for the phage T7, T3 and SP6 are quite similar, each beingabout 15 to 20 basepairs long, and sharing about 45% identity between−17 and −1 nucleotides, relative to the start of transcription. Despitethese similarities, the RNA polymerases from these phage are highlyspecific for their cognate promoters, such that the other promoters maybe present in a reaction, but will not be transcribed (Chamberlin andRyan, Enzymes XV:87-108 [1982]). Because these promoters are similar insize and in the way in which they are recognized by their polymerases(Li et al., Biochem. 35:3722 [1996]) similar nicked versions of thepromoters may be designed for use in the methods of the presentinvention by analogy to the examples described herein which employ theT7 promoter. Because of the high degree of specificity of the RNApolymerases, these nicked promoters may be used together to detectmultiple targets in a single reaction. There are many instances in whichit would be highly desirable to detect multiple nucleic acid targets ina single sample, including cases in which multiple infectious agents maybe present, or in which variants of a single type of target may need tobe identified. Alternatively, it is often desirable to use a combinationof probes to detect both a target sequence and an internal controlsequence, to gauge the effects of sample contaminants on the output ofthe assay. The use of multiple promoters allows the reaction to beassessed for both the efficiency of the invasive cleavage and therobustness of the transcription.

As stated above, the phage promoters were described in detail as anexample of suitable protein binding regions (e.g., which can be used togenerate a composite promoter) for use in the methods of the presentinvention. The invention is not limited to the use of phage RNApolymerase promoter regions, in particular, and RNA polymerase promoterregions, in general. Suitably specific, well characterized promoters arealso found in both prokaryotic and eukaryotic systems.

The RNA that is produced in a manner that is dependent of the successfuldetection of the target nucleic acid in the invasive cleavage reactionmay be detected in any of several ways. If a labeled nucleotide isincorporated into the RNA during transcription, the RNA may be detecteddirectly after fractionation (e.g., by electrophoresis orchromatography). The labeled RNA may also be captured onto a solidsupport, such as a microtitre plate, a bead or a dipstick (e.g., byhybridization, antibody capture, or through an affinity interaction suchas that between biotin and avidin). Capture may facilitate the measuringof incorporated label, or it may be an intermediate step before probehybridization or similar detection means. If the maximum amount of labelis desired to be incorporated into each transcript, it is preferred thatthe copy template be very long, around 3 to 10 kilobases, so that eachRNA molecule will carry many labels. Alternatively, it may be desiredthat a single site or a limited number of sites within the transcript bespecifically labeled. In this case, it may be desirable to have a shortcopy template with only one or a few residues that would allowincorporation of the labeled nucleotide.

The copy template may also be selected to produce RNAs that performspecified functions. In a simple case, if an duplex-dependentintercalating fluorophore is to be used to detect the RNA product, itmay be desirable to transcribe an RNA that is known to form duplexedsecondary structures, such as a ribosomal RNA or a tRNA. In anotherembodiment, the RNA may be designed to interact specifically, or withparticular affinity, with a different substance. It has been shown thata process of alternating steps of selection (e.g., by binding to atarget substance) and in vitro amplification (e.g., by PCR) can be usedto identify nucleic acid ligands with novel and useful properties (Tuerkand Gold, Science 249:505 [1990]). This system has been used to identifyRNAs, termed ligands or aptamers, that bind tightly and specifically toproteins and to other types of molecules, such as antibiotics (Wang etal., Biochem. 35:12338 [1996]) and hormones. RNAs can even be selectedto bind to other RNAs through non-Watson-Crick interactions (Schmidt etal., Ann. N.Y. Acad. Sci. 782:526 [1996]). A ligand RNA may be used toeither inactivate or enhance the activity of a molecule to which itbinds. Any RNA segment identified through such a process may also beproduced by the methods of the present invention, so that theobservation of the activity of the RNA ligand may be used as a specificsign of the presence of the target material in the invasive cleavagereaction. The ligand binding to its specific partner may also be used asanother way of capturing a readout signal to a solid support.

The product RNA might also be designed to have a catalytic function(e.g., to act as a ribozyme), allowing cleavage another molecule to beindicative of the success of the primary invasive cleavage reaction(Uhlenbeck, Nature 328:596 [1987]). In yet another embodiment, the RNAmay be made to encode a peptide sequence. When coupled to an in vitrotranslation system (e.g., the S-30 system derived from E. coli [Lesley,Methods Mol. Biol., 37:265 (1985)], or a rabbit reticulocyte lysatesystem [Dasso and Jackson, Nucleic Acids Res. 17:3129 (1989)], availablefrom Promega), the production of the appropriate protein may bedetected. In a preferred embodiment, the proteins produced include thosethat allow either colorimetric or luminescent detection, such asbeta-galactosidase (lac-Z) or luciferase, respectively.

The above discussion focused on the use of the present transcriptionvisualization methods in the context of the INVADER-directed cleavageassay (i.e., the non-target cleavage products produced in the INVADERassay were used to complete and activate a protein binding region, suchas a promoter region). However, the transcription visualization methodsare not limited to this context. Any assay that produces anoligonucleotide product having relatively discrete ends can be used inconjunction with the present transcription visualization methods. Forexample, the homogenous assay described in U.S. Pat. No. 5,210,015,particularly when conducted under conditions where polymerization cannotoccur, produces short oligonucleotide fragments as the result ofcleavage of a probe. If this assay is conducted under conditions wherepolymerization occurs, the site of cleavage of the probe may be focusedthrough the use of nucleotide analogs that have uncleavable linkages atparticular positions within the probe. These short oligonucleotides canbe employed in a manner analogous to the cut probe or non-targetcleavage products produced in the invasive cleavage reactions of thepresent invention. Additional assays that generate suitableoligonucleotide products are known to the art. For example, thenon-target cleavage products produced in assays such as the “CyclingProbe Reaction” (Duck et al., BioTech., 9:142 [1990] and U.S. Pat. Nos.4,876,187 and 5,011,769, herein incorporated by reference), in whichshorter oligonucleotides are released from longer oligonucleotides afterhybridization to a target sequence would be suitable, as would shortrestriction fragments released in assays where a probe is designed to becleaved when successfully hybridized to an appropriate restrictionrecognition sequence (U.S. Pat. No. 4,683,194, herein incorporated byreference).

Assays that generate short oligonucleotides having “ragged” (i.e., notdiscrete) 3′ ends can also be employed with success in the transcriptionreactions of the present invention when the oligonucleotide provided bythis non-transcription reaction are used to provide a portion of thepromoter region located downstream of the other oligonucleotide(s) thatare required to complete the promoter region (that is a 3′ tail orunpaired extension can be tolerated when the oligonucleotide is beingused as the “Cut Probe” is in FIGS. 94 and 95A).

VII. Generation of 5′ Nucleases Derived from Thermostable DNAPolymerases

The 5′ nucleases of the invention form the basis of a novel detectionassay for the identification of specific nucleic acid sequences. FIG. 1Aprovides a schematic of one embodiment of the detection method of thepresent invention. The target sequence is recognized by two distinctoligonucleotides in the triggering or trigger reaction. In a preferredembodiment, one of these oligonucleotides is provided on a solidsupport. The other can be provided free in solution. In FIG. 1A the freeoligonucleotide is indicated as a “primer” and the other oligonucleotideis shown attached to a bead designated as type 1. The target nucleicacid aligns the two oligonucleotides for specific cleavage of the 5′ arm(of the oligonucleotide on bead 1) by the 5′ nucleases of the presentinvention (not shown in FIG. 1A). The site of cleavage (indicated by alarge solid arrowhead) is controlled by the position of the 3′ end ofthe “primer” relative to the downstream fork of the oligonucleotide onbead 1.

Successful cleavage releases a single copy of what is referred to as thealpha signal oligonucleotide. This oligonucleotide may contain adetectable moiety (e.g., fluorescein). On the other hand, it may beunlabeled.

In one embodiment of the detection method, two more oligonucleotides areprovided on solid supports. The oligonucleotide shown in FIG. 1A on bead2 has a region that is complementary to the alpha signal oligonucleotide(indicated as alpha prime) allowing for hybridization. This structurecan be cleaved by the 5′ nucleases of the present invention to releasethe beta signal oligonucleotide. The beta signal oligonucleotide canthen hybridize to type 3 beads having an oligonucleotide with acomplementary region (indicated as beta prime). Again, this structurecan be cleaved by the 5′ nucleases of the present invention to release anew alpha oligonucleotide.

Up to this point, the amplification has been linear. To increase thepower of the method, it is desired that the alpha signal oligonucleotidehybridized to bead type 2 be liberated after release of the betaoligonucleotide so that it may go on to hybridize with otheroligonucleotides on type 2 beads. Similarly, after release of an alphaoligonucleotide from type 3 beads, it is desired that the betaoligonucleotide be liberated.

With the liberation of signal oligonucleotides by such techniques, eachcleavage results in a doubling of the number of signal oligonucleotides.In this manner, detectable signal can quickly be achieved.

FIG. 1B provides a schematic of a second embodiment of the detectionmethod of the present invention. Again, the target sequence isrecognized by two distinct oligonucleotides in the triggering or triggerreaction and the target nucleic acid aligns the two oligonucleotides forspecific cleavage of the 5′ arm by the DNAPs of the present invention(not shown in FIG. 1B). In this specific example, the firstoligonucleotide is completely complementary to a portion of the targetsequence. The second oligonucleotide is partially complementary to thetarget sequence; the 3′ end of the second oligonucleotide is fullycomplementary to the target sequence while the 5′ end isnon-complementary and forms a single-stranded arm. The non-complementaryend of the second oligonucleotide may be a generic sequence that can beused with a set of standard hairpin structures (described below). Thedetection of different target sequences would require unique portions oftwo oligonucleotides: the entire first oligonucleotide and the 3′ end ofthe second oligonucleotide. The 5′ arm of the second oligonucleotide canbe invariant or generic in sequence.

The second part of the detection method allows the annealing of thefragment of the second oligonucleotide liberated by the cleavage of thefirst cleavage structure formed in the triggering reaction (called thethird or trigger oligonucleotide) to a first hairpin structure. Thisfirst hairpin structure has a single-stranded 5′ arm and asingle-stranded 3′ arm. The third oligonucleotide triggers the cleavageof this first hairpin structure by annealing to the 3′ arm of thehairpin thereby forming a substrate for cleavage by the 5′ nuclease ofthe present invention. The cleavage of this first hairpin structuregenerates two reaction products: 1) the cleaved 5′ arm of the hairpincalled the fourth oligonucleotide, and 2) the cleaved hairpin structurethat now lacks the 5′ arm and is smaller in size than the uncleavedhairpin. This cleaved first hairpin may be used as a detection moleculeto indicate that cleavage directed by the trigger or thirdoligonucleotide occurred. Thus, this indicates that the first twooligonucleotides found and annealed to the target sequence therebyindicating the presence of the target sequence in the sample.

The detection products may be amplified by having the fourtholigonucleotide anneal to a second hairpin structure. This hairpinstructure has a 5′ single-stranded arm and a 3′ single-stranded arm. Thefourth oligonucleotide generated by cleavage of the first hairpinstructure anneals to the 3′ arm of the second hairpin structure therebycreating a third cleavage structure recognized by the 5′ nuclease. Thecleavage of this second hairpin structure also generates two reactionproducts: 1) the cleaved 5′ arm of the hairpin called the fiftholigonucleotide, and 2) the cleaved second hairpin structure which nowlacks the 5′ arm and is smaller in size than the uncleaved hairpin. Inone embodiment, the fifth oligonucleotide is similar or identical insequence to the third nucleotide. The cleaved second hairpin may beviewed as a detection molecule that amplifies the signal generated bythe cleavage of the first hairpin structure. Simultaneously with theannealing of the forth oligonucleotide, the third oligonucleotide isdissociated from the cleaved first hairpin molecule so that it is freeto anneal to a new copy of the first hairpin structure. Thedisassociation of the oligonucleotides from the hairpin structures maybe accomplished by heating or other means suitable to disruptbase-pairing interactions. As described above, conditions may beselected that allow the association and disassociation of hybridizedoligonucleotides without temperature cycling.

If fifth oligonucleotide is similar or identical in sequence to thethird oligonucleotide, further amplification of the detection signal isachieved by annealing the fifth oligonucleotide to another molecule ofthe first hairpin structure. Cleavage is then performed and theoligonucleotide that is liberated then is annealed to another moleculeof the second hairpin structure. Successive rounds of annealing andcleavage of the first and second hairpin structures, provided in excess,are performed to generate a sufficient amount of cleaved hairpinproducts to be detected.

As discussed above for other embodiments of detection usingstructure-specific nuclease cleavage, any method known in the art foranalysis of nucleic acids, nucleic acid fragments or oligonucleotidesmay be applied to the detection of these cleavage products.

The hairpin structures may be attached to a solid support, such as anagarose, styrene or magnetic bead, via the 3′ end of the hairpin. Aspacer molecule may be placed between the 3′ end of the hairpin and thebead, if so desired. The advantage of attaching the hairpin structuresto a solid support is that this prevents the hybridization of the twohairpin structures to one another over regions which are complementary.If the hairpin structures anneal to one another, this would reduce theamount of hairpins available for hybridization to the primers releasedduring the cleavage reactions. If the hairpin structures are attached toa solid support, then additional methods of detection of the products ofthe cleavage reaction may be employed. These methods include, but arenot limited to, the measurement of the released single-stranded 5′ armwhen the 5′ arm contains a label at the 5′ terminus. This label may beradioactive, fluorescent, biotinylated, etc. If the hairpin structure isnot cleaved, the 5′ label will remain attached to the solid support. Ifcleavage occurs, the 5′ label will be released from the solid support.

The 3′ end of the hairpin molecule may be blocked through the use ofdideoxynucleotides. A 3′ terminus containing a dideoxynucleotide isunavailable to participate in reactions with certain DNA modifyingenzymes, such as terminal transferase. Cleavage of the hairpin having a3′ terminal dideoxynucleotide generates a new, unblocked 3′ terminus atthe site of cleavage. This new 3′ end has a free hydroxyl group that caninteract with terminal transferase thus providing another means ofdetecting the cleavage products.

The hairpin structures are designed so that their self-complementaryregions are very short (generally in the range of 3-8 base pairs). Thus,the hairpin structures are not stable at the high temperatures at whichthis reaction is performed (generally in the range of 50-75° C.) unlessthe hairpin is stabilized by the presence of the annealedoligonucleotide on the 3′ arm of the hairpin. This instability preventsthe polymerase from cleaving the hairpin structure in the absence of anassociated primer thereby preventing false positive results due tonon-oligonucleotide directed cleavage.

VIII. Improved Enzymes for Use in INVADER Oligonucleotide-DirectedCleavage Reactions

A cleavage structure is defined herein as a structure that is formed bythe interaction of a probe oligonucleotide and a target nucleic acid toform a duplex, the resulting structure being cleavable by a cleavageagent, including but not limited to an enzyme. The cleavage structure isfurther defined as a substrate for specific cleavage by the cleavagemeans in contrast to a nucleic acid molecule that is a substrate fornonspecific cleavage by agents such as phosphodiesterases. Examples ofsome possible cleavage structures are shown in FIG. 15. In consideringimprovements to enzymatic cleavage agents, one may consider the actionof said enzymes on any of these structures, and on any other structuresthat fall within the definition of a cleavage structure. The cleavagesites indicated on the structures in FIG. 15 are presented by way ofexample. Specific cleavage at any site within such a structure iscontemplated.

Improvements in an enzyme may be an increased or decreased rate ofcleavage of one or more types of structures. Improvements may alsoresult in more or fewer sites of cleavage on one or more of saidcleavage structures. In developing a library of new structure-specificnucleases for use in nucleic acid cleavage assays, improvements may havemany different embodiments, each related to the specific substratestructure used in a particular assay.

As an example, one embodiment of the INVADER oligonucleotide-directedcleavage assay of the present invention may be considered. In theINVADER oligonucleotide-directed cleavage assay, the accumulation ofcleaved material is influenced by several features of the enzymebehavior. Not surprisingly, the turnover rate, or the number ofstructures that can be cleaved by a single enzyme molecule in a setamount of time, is very important in determining the amount of materialprocessed during the course of an assay reaction. If an enzyme takes along time to recognize a substrate (e.g., if it is presented with aless-than-optimal structure), or if it takes a long time to executecleavage, the rate of product accumulation is lower than if these stepsproceeded quickly. If these steps are quick, yet the enzyme “holds on”to the cleaved structure, and does not immediately proceed to anotheruncut structure, the rate will be negatively affected.

Enzyme turnover is not the only way in which enzyme behavior cannegatively affect the rate of accumulation of product. When the meansused to visualize or measure product is specific for a precisely definedproduct, products that deviate from that definition may escapedetection, and thus the rate of product accumulation may appear to belower than it is. For example, if one had a sensitive detector fortrinucleotides that could not see di- or tetranucleotides, or any sizedoligonucleotide other that 3 residues, in the INVADER-directed cleavageassay of the present invention any errant cleavage would reduce thedetectable signal proportionally. It can be seen from the cleavage datapresented here that, while there is usually one site within a probe thatis favored for cleavage, there are often products that arise fromcleavage one or more nucleotides away from the primary cleavage site.These are products that are target-dependent, and are thus notnon-specific background. Nevertheless, if a subsequent visualizationsystem can detect only the primary product, these represent a loss ofsignal. One example of such a selective visualization system is thecharge reversal readout presented herein, in which the balance ofpositive and negative charges determines the behavior of the products.In such a system the presence of an extra nucleotide or the absence ofan expected nucleotide can excluded a legitimate cleavage product fromultimate detection by leaving that product with the wrong balance ofcharge. It can be easily seen that any assay that can sensitivelydistinguish the nucleotide content of an oligonucleotide, such asstandard stringent hybridization, suffers in sensitivity when somefraction of the legitimate product is not eligible for successfuldetection by that assay.

These discussions suggest two highly desirable traits in any enzyme tobe used in the method of the present invention. First, the more rapidlythe enzyme executes an entire cleavage reaction, including recognition,cleavage and release, the more signal it may potentially created in theINVADER oligonucleotide-directed cleavage assay. Second, the moresuccessful an enzyme is at focusing on a single cleavage site within astructure, the more of the cleavage product can be successfully detectedin a selective read-out.

The rationale cited above for making improvements in enzymes to be usedin the INVADER oligonucleotide-directed cleavage assay are meant toserve as an example of one direction in which improvements might besought, but not as a limit on either the nature or the applications ofimproved enzyme activities. As another direction of activity change thatwould be appropriately considered improvement, the DNAP-associated 5′nucleases may be used as an example. In creating some of thepolymerase-deficient 5′ nucleases described herein it was found that thethose that were created by deletion of substantial portions of thepolymerase domain, as depicted in FIG. 4, assumed activities that wereweak or absent in the parent proteins. These activities included theability to cleave the non-forked structure shown in FIG. 15D, a greatlyenhanced ability to exonucleolytically remove nucleotides from the 5′ends of duplexed strands, and a nascent ability to cleave circularmolecules without benefit of a free 5′ end.

In addition to the 5′ nucleases derived from DNA polymerases, thepresent invention also contemplates the use of structure-specificnucleases that are not derived from DNA polymerases. For example, aclass of eukaryotic and archaebacterial endonucleases have beenidentified which have a similar substrate specificity to 5′ nucleases ofPol I-type DNA polymerases. These are the FEN1 (Flap EndoNuclease),RAD2, and XPG (Xeroderma Pigmentosa-complementation group G) proteins.These proteins are involved in DNA repair, and have been shown to favorthe cleavage of structures that resemble a 5′ arm that has beendisplaced by an extending primer during polymerization, similar to themodel depicted in FIG. 15B. Similar DNA repair enzymes have beenisolated from single cell and higher eukaryotes and from archaea, andthere are related DNA repair proteins in eubacteria. Similar 5′nucleases have also been associated with bacteriophage such as T5 andT7.

Recently, the 3-dimensional structures of DNAPTaq and T5 phage5′-exonuclease (FIG. 58) were determined by X-ray diffraction (Kim etal., Nature 376:612 [1995]; and Ceska et al., Nature 382:90 [1995]). Thetwo enzymes have very similar 3-dimensional structures despite limitedamino acid sequence similarity. The most striking feature of the T55′-exonuclease structure is the existence of a triangular hole formed bythe active site of the protein and two alpha helices (FIG. 58). Thissame region of DNAPTaq is disordered in the crystal structure,indicating that this region is flexible, and thus is not shown in thepublished 3-dimensional structure. However, the 5′ nuclease domain ofDNAPTaq is likely to have the same structure, based its overall3-dimensional similarity to T5 5′-exonuclease, and that the amino acidsin the disordered region of the DNAPTaq protein are those associatedwith alpha helix formation. The existence of such a hole or groove inthe 5′ nuclease domain of DNAPTaq was predicted based on its substratespecificity (Lyamichev et al., supra).

It has been suggested that the 5′ arm of a cleavage structure mustthread through the helical arch described above to position saidstructure correctly for cleavage (Ceska et al., supra). One of themodifications of 5′ nucleases described herein opened up the helicalarch portion of the protein to allow improved cleavage of structuresthat cut poorly or not at all (e.g., structures on circular DNA targetsthat would preclude such threading of a 5′ arm). The gene construct thatwas chosen as a model to test this approach was the one called CLEAVASEBN, which was derived from DNAPTaq but does not contain the polymerasedomain (Ex. 2). It comprises the entire 5′ nuclease domain of DNAP Taq,and thus should be very close in structure to the T5 5′ exonuclease.This 5′ nuclease was chosen to demonstrate the principle of such aphysical modification on proteins of this type. The arch-openingmodification of the present invention is not intended to be limited tothe 5′ nuclease domains of DNA polymerases, and is contemplated for useon any structure-specific nuclease that includes such an aperture as alimitation on cleavage activity. The present invention contemplates theinsertion of a thrombin cleavage site into the helical arch of DNAPsderived from the genus Thermus as well as 5′ nucleases derived fromDNAPs derived from the genus Thermus. The specific example shown hereinusing the CLEAVASE BN/thrombin nuclease merely illustrates the conceptof opening the helical arch located within a nuclease domain. As theamino acid sequence of DNAPs derived from the genus Thermus are highlyconserved, the teachings of the present invention enable the insertionof a thrombin site into the helical arch present in these DNAPs and 5′nucleases derived from these DNAPs.

The opening of the helical arch was accomplished by insertion of aprotease site in the arch. This allowed post-translational digestion ofthe expressed protein with the appropriate protease to open the arch atits apex. Proteases of this type recognize short stretches of specificamino acid sequence. Such proteases include thrombin and factor Xa.Cleavage of a protein with such a protease depends on both the presenceof that site in the amino acid sequence of the protein and theaccessibility of that site on the folded intact protein. Even with acrystal structure it can be difficult to predict the susceptibility ofany particular region of a protein to protease cleavage. Absent acrystal structure it must be determined empirically.

In selecting a protease for a site-specific cleavage of a protein thathas been modified to contain a protease cleavage site, a first step isto test the unmodified protein for cleavage at alternative sites. Forexample, DNAPTaq and CLEAVASE BN nuclease were both incubated underprotease cleavage conditions with factor Xa and thrombin proteases. Bothnuclease proteins were cut with factor Xa within the 5′ nuclease domain,but neither nuclease was digested with large amounts of thrombin. Thus,thrombin was chosen for initial tests on opening the arch of theCLEAVASE BN enzyme.

In the protease/CLEAVASE modifications described herein the factor Xaprotease cleaved strongly in an unacceptable position in the unmodifiednuclease protein, in a region likely to compromise the activity of theend product. Other unmodified nucleases contemplated herein may not besensitive to the factor Xa, but may be sensitive to thrombin or othersuch proteases. Alternatively, they may be sensitive to these or othersuch proteases at sites that are immaterial to the function of thenuclease sought to be modified. In approaching any protein formodification by addition of a protease cleavage site, the unmodifiedprotein should be tested with the proteases under consideration todetermine which proteases give acceptable levels of cleavage in otherregions.

Working with the cloned segment of DNAPTaq from which the CLEAVASE BNprotein is expressed, nucleotides encoding a thrombin cleavage site wereintroduced in-frame near the sequence encoding amino acid 90 of thenuclease gene. This position was determined to be at or near the apex ofthe helical arch by reference to both the 3-dimensional structure ofDNAPTaq, and the structure of T5 5′ exonuclease. The encoded amino acidsequence, LVPRGS, was inserted into the apex of the helical arch bysite-directed mutagenesis of the nuclease gene. The proline (P) in thethrombin cleavage site was positioned to replace a proline normally inthis position in CLEAVASE BN because proline is an alpha helix-breakingamino acid, and may be important for the 3-dimensional structure of thisarch. This construct was expressed, purified and then digested withthrombin. The digested enzyme was tested for its ability to cleave atarget nucleic acid, bacteriophage M13 genomic DNA, that does notprovide free 5′ ends to facilitate cleavage by the threading model.

While the helical arch in this nuclease was opened by protease cleavage,it is contemplated that a number of other techniques could be used toachieve the same end. For example, the nucleotide sequence could berearranged such that, upon expression, the resulting protein would beconfigured so that the top of the helical arch (amino acid 90) would beat the amino terminus of the protein, the natural carboxyl and aminotermini of the protein sequence would be joined, and the new carboxylterminus would lie at natural amino acid 89. This approach has thebenefit that no foreign sequences are introduced and the enzyme is asingle amino acid chain, and thus may be more stable that the cleaved 5′nuclease. In the crystal structure of DNAPTaq, the amino and carboxyltermini of the 5′-exonuclease domain lie in close proximity to eachother, which suggests that the ends may be directly joined without theuse of a flexible linker peptide sequence as is sometimes necessary.Such a rearrangement of the gene, with subsequent cloning and expressioncould be accomplished by standard PCR recombination and cloningtechniques known to those skilled in the art.

The present invention also contemplates the use of nucleases isolatedfrom organisms that grow under a variety of conditions. The genes forthe FEN-1/XPG class of enzymes are found in organisms ranging frombacteriophage to humans to the extreme thermophiles of Kingdom Archaea.For assays in which high temperature is to be used, it is contemplatedthat enzymes isolated from extreme thermophiles may exhibit thethermostability required of such an assay. For assays in which it mightbe desirable to have peak enzyme activity at moderate temperature or inwhich it might be desirable to destroy the enzyme with elevatedtemperature, those enzymes from organisms that favor moderatetemperatures for growth may be of particular value.

An alignment of a collection of FEN-1 proteins sequenced by others isshown in FIGS. 59A-E (SEQ ID NOS:135-145). It can be seen from thisalignment that there are some regions of conservation in this class ofproteins, suggesting that they are related in function, and possibly instructure. Regions of similarity at the amino acid sequence level can beused to design primers for in vitro amplification (PCR) by a process ofback translating the amino acid sequence to the possible nucleic acidsequences, then choosing primers with the fewest possible variationswithin the sequences. These can be used in low stringency PCR to searchfor related DNA sequences. This approach permits the amplification ofDNA encoding a FEN-1 nuclease without advance knowledge of the actualDNA sequence.

It can also be seen from this alignment that there are regions in thesequences that are not completely conserved. The degree of differenceobserved suggests that the proteins may have subtle or distinctdifferences in substrate specificity. In other words, they may havedifferent levels of cleavage activity on the cleavage structures of thepresent invention. When a particular structure is cleaved at a higherrate than the others, this is referred to a preferred substrate, while astructure that is cleaved slowly is considered a less preferredsubstrate. The designation of preferred or less preferred substrates inthis context is not intended to be a limitation of the presentinvention. It is contemplated that some embodiments the presentinvention will make use of the interactions of an enzyme with a lesspreferred substrate. Candidate enzymes are tested for suitability in thecleavage assays of the present invention using the assays describedbelow.

1. Structure Specific Nuclease Assay

Testing candidate nucleases for structure-specific activities in theseassays is done in much the same way as described for testing modifiedDNA polymerases in Example 2, but with the use of a different library ofmodel structures. In addition to assessing the enzyme performance inprimer-independent and primer-directed cleavage, a set of synthetichairpins are used to examine the length of duplex downstream of thecleavage site preferred by the enzyme.

The FEN-1 and XPG 5′ nucleases used in the present invention should betested for activity in the assays in which they are intended to be used,including but not limited to the INVADER-directed cleavage detectionassay of the present invention and the CFLP method of characterizingnucleic acids (the CFLP method is described in U.S. Pat. Nos. 5,843,654,5,843,669, 5,719,028, and 5,888,780 and PCT Publication WO 96/15267; thedisclosures of which are incorporated herein by reference). The INVADERassay uses a mode of cleavage that has been termed “primer directed” of“primer dependent” to reflect the influence of the an oligonucleotidehybridized to the target nucleic acid upstream of the cleavage site. Incontrast, the CFLP reaction is based on the cleavage of foldedstructure, or hairpins, within the target nucleic acid, in the absenceof any hybridized oligonucleotide. The tests described herein are notintended to be limited to the analysis of nucleases with any particularsite of cleavage or mode of recognition of substrate structures. It iscontemplated that enzymes may be described as 3′ nucleases, utilizingthe 3′ end as a reference point to recognize structures, or may have ayet a different mode of recognition. Further, the use of the term 5′nucleases is not intended to limit consideration to enzymes that cleavethe cleavage structures at any particular site. It refers to a generalclass of enzymes that require some reference or access to a 5′ end toeffect cleavage of a structure.

A set of model cleavage structures has been created to allow thecleavage ability of unknown enzymes on such structures to be assessed.Each of the model structures is constructed of one or more syntheticoligonucleotides made by standard DNA synthesis chemistry. Examples ofsuch synthetic model substrate structures are shown in FIGS. 26 and 60.These are intended only to represent the general folded configurationdesirable is such test structures. While a sequence that would assumesuch a structure is indicated in the Figures, there are numerous othersequence arrangements of nucleotides that would be expected to fold insuch ways. The essential features to be designed into a set ofoligonucleotides to perform the tests described herein are the presenceor absence of a sufficiently long 3′ arm to allow hybridization of anadditional nucleic acid to test cleavage in a “primer-directed” mode,and the length of the duplex region. In the set depicted in FIG. 60, theduplex lengths of the S-33 and the 11-8-0 structures are 12 and 8basepairs, respectively. This difference in length in the test moleculesfacilitates detection of discrimination by the candidate nucleasebetween longer and shorter duplexes. Additions to this series expandingthe range of duplex molecules presented to the enzymes, both shorter andlonger, may be used. The use of a stabilizing DNA tetraloop (Antao etal., Nucl. Acids Res., 19:5901 [1991]) or triloop (Hiraro et al., Nuc.Acids Res., 22:576 [1994]) at the closed end of the duplex helps ensureformation of the expected structure by the oligonucleotide.

The model substrate for testing primer directed cleavage, the “S-60hairpin” (SEQ ID NO:40) is described in Example 11. In the absence of aprimer this hairpin is usually cleaved to release 5′ arm fragments of 18and 19 nucleotides length. An oligonucleotide, termed P-14(5′-CGAGAGACCACGCT-3′; SEQ ID NO:108), that extends to the base of theduplex when hybridized to the 3′ arm of the S-60 hairpin gives cleavageproducts of the same size, but at a higher rate of cleavage.

To test invasive cleavage a different primer is used, termed P-15(5′-CGAGAGACCACGCTG-3′; SEQ ID NO:30). In a successful invasive cleavagethe presence of this primer shifts the site of cleavage of S-60 into theduplex region, usually releasing products of 21 and 22 nucleotideslength.

The S-60 hairpin may also be used to test the effects of modificationsof the cleavage structure on either primer-directed or invasivecleavage. Such modifications include, but are not limited to, use ofmismatches or base analogs in the hairpin duplex at one, a few or allpositions, similar disruptions or modifications in the duplex betweenthe primer and the 3′ arm of the S-60, chemical or other modificationsto one or both ends of the primer sequence, or attachment of moietiesto, or other modifications of the 5′ arm of the structure. In all of theanalyses using the S-60 or a similar hairpin described herein, activitywith and without a primer may be compared using the same hairpinstructure.

The assembly of these test reactions, including appropriate amounts ofhairpin, primer and candidate nuclease is described in Example 2. Ascited therein, the presence of cleavage products is indicated by thepresence of molecules that migrate at a lower molecular weight than doesthe uncleaved test structure. When the reversal of charge of a label isused the products will carry a different net charge than the uncleavedmaterial. Any of these cleavage products indicate that the candidatenuclease has the desired structure-specific nuclease activity. By“desired structure-specific nuclease activity” it is meant only that thecandidate nuclease cleaves one or more test molecules. It is notnecessary that the candidate nuclease cleave at any particular rate orsite of cleavage to be considered successful cleavage.

2. Enzyme Chimeras and Variants

The present invention further provides chimerical structure-specificnucleases. Chimerical structure-specific nucleases comprise one or moreportions of any of the enzymes described herein in combination withanother sequence. In preferred embodiments, the chimericalstructure-specific nucleases comprise a functional domain (e.g., aregion of the enzyme containing an arch region or sequence physicallyassociated therewith) from a 5′-nuclease in combination with domainsfrom other enzymes (e.g., from other 5′-nucleases). In some preferredembodiments, a given functional domain comprises sequence from two ormore enzymes. For example, the amino acid sequence of a functionaldomain of a first structure-specific nuclease may be altered at one ormore amino acid positions to convert the functional domain, or a portionthereof, to the sequence of a second structure-specific nuclease,thereby imparting characteristics of the second nuclease on the first.Such characteristics include, but are not limited to catalytic activity,specificity, and stability (e.g., thermostability).

In one embodiment, the present invention provides chimerical enzymescomprising amino acid portions derived from the enzymes selected fromthe group of DNA polymerases and FEN-1, XPG and RAD endonucleases. In apreferred embodiment, the chimerical enzymes comprise amino acidportions derived from the FEN-1 endonucleases selected from the group ofPyrococcus furiosus, Methanococcus jannaschi, Pyrococcus woesei,Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, Sulfolobussolfataricus, Pyrobaculum aerophilum, Thermococcus litoralis,Archaeaglobus veneficus, Archaeaglobus profundus, Acidianus brierlyi,Acidianus ambivalens, Desulfurococcus amylolyticus, Desulfurococcusmobilis, Pyrodictium brockii, Thermococcus gorgonarius, Thermococcuszilligii, Methanopyrus kandleri, Methanococcus igneus, Pyrococcushorikoshii, and Aeropyrum pernix.

Some embodiments of the present invention provide mutant or variantforms of enzymes described herein. It is possible to modify thestructure of a peptide having an activity of the enzymes describedherein for such purposes as enhancing cleavage rate, substratespecificity, stability, and the like. For example, a modified peptidecan be produced in which the amino acid sequence has been altered, suchas by amino acid substitution, deletion, or addition. For example, it iscontemplated that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid (i.e., conservative mutations) will not have a majoreffect on the biological activity of the resulting molecule.Accordingly, some embodiments of the present invention provide variantsof enzymes described herein containing conservative replacements.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Genetically encodedamino acids can be divided into four families: (1) acidic (aspartate,glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan); and (4) uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. In similar fashion, the amino acid repertoire can begrouped as (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine histidine), (3) aliphatic (glycine, alanine, valine, leucine,isoleucine, serine, threonine), with serine and threonine optionally begrouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine,tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (See e.g., Stryer (ed.),Biochemistry, 2nd ed, WH Freeman and Co. [1981]). Whether a change inthe amino acid sequence of a peptide results in a functional homolog canbe readily determined by assessing the ability of the variant peptide toproduce a response in a fashion similar to the wild-type protein usingthe assays described herein. Peptides in which more than one replacementhas taken place can readily be tested in the same manner.

It is contemplated that the nucleic acids encoding the enzymes can beutilized as starting nucleic acids for directed evolution. Thesetechniques can be utilized to develop enzyme variants having desirableproperties. In some embodiments, artificial evolution is performed byrandom mutagenesis (e.g., by utilizing error-prone PCR to introducerandom mutations into a given coding sequence). This method requiresthat the frequency of mutation be finely tuned. As a general rule,beneficial mutations are rare, while deleterious mutations are common.This is because the combination of a deleterious mutation and abeneficial mutation often results in an inactive enzyme. The idealnumber of base substitutions for targeted gene is usually between 1.5and 5 (Moore and Arnold, Nat. Biotech., 14, 458-67 [1996]; Leung et al.,Technique, 1:11-15 [1989]; Eckert and Kunkel, PCR Methods Appl., 1:17-24[1991]; Caldwell and Joyce, PCR Methods Appl., 2:28-33 (1992); and Zhaoand Arnold, Nuc. Acids. Res., 25:1307-08 [1997]). After mutagenesis, theresulting clones are selected for desirable activity (e.g., ability tocleave a cleavage structure such as those described in Example 66).Successive rounds of mutagenesis and selection are often necessary todevelop enzymes with desirable properties. It should be noted that onlythe useful mutations are carried over to the next round of mutagenesis.

In other embodiments of the present invention, the polynucleotides ofthe present invention are used in gene shuffling or sexual PCRprocedures (e.g., Smith, Nature, 370:324-25 [1994]; U.S. Pat. Nos.5,837,458; 5,830,721; 5,811,238; 5,733,731; all of which are hereinincorporated by reference). Gene shuffling involves random fragmentationof several mutant DNAs followed by their reassembly by PCR into fulllength molecules. Examples of various gene shuffling procedures include,but are not limited to, assembly following DNase treatment, thestaggered extension process (STEP), and random priming in vitrorecombination. In the DNase mediated method, DNA segments isolated froma pool of positive mutants are cleaved into random fragments with DNaseIand subjected to multiple rounds of PCR with no added primer. Thelengths of random fragments approach that of the uncleaved segment asthe PCR cycles proceed, resulting in mutations in different clonesbecoming mixed and accumulating in some of the resulting sequences.Multiple cycles of selection and shuffling have led to the functionalenhancement of a number of enzymes (Stemmer, Nature, 370:398-91 [1994];Stemmer, Proc. Natl. Acad. Sci. USA, 91, 10747-51 [1994]; Crameri etal., Nat. Biotech., 14:315-19 [1996]; Zhang et al., Proc. Natl. Acad.Sci. USA, 94:4504-09 [1997]; and Crameri et al., Nat. Biotech.,15:436-38 [1997]).

The present invention provides a means of rapidly screening enzymes forimproved activity. In some embodiments, the rapid screening method ofthe present invention comprises an instrument system comprising a liquidhandling function (e.g., a BIOMEK 2000 Laboratory Automated workstation, Beckman Coulter, Fullerton Calif., or TECAN Automatedworkstation, Tecan U.S., Durham N.C.), a heating block function, anincubator function (e.g., a Liconic Instruments Automated Incubator,Liconic Instruments, Fusrentum, Liechtenstein or a HERAEUS automatedincubator), a microplate carousel function (e.g., a BIOMEK Carousel,Beckman Coulter, Fullerton, Calif.), a fluorescence reader function(e.g., a CYTOFLUOR Series 4000 multiwell plate reader or a Sapphireautomated plate reader (Tecan, U.S.) and a Robotic function (e.g., aBIOMEK ORCA robot arm, Beckman Coulter). An exemplary diagram of thescreening system of the present invention is provided in FIG. 147. Inone embodiment of the screening method of the present invention, therobot function moves pipet tips, lysate plates (e.g., 96-well), platesof cultures (e.g., Deep Well Growth Plate filled with mutated culture)with the ORCA arm from the Carousel to the BIOMEK. The BIOMEK thendispenses lysozyme mix to each well of the lysate plates. Cell cultureis then transferred from each well of the plate of culture to eachcorresponding well of the lysate plate. The lysate plates are thentransferred by the ORCA arm to the incubator for a period of time (e.g.,room temperature for 15 minutes), then transferred to the heating blockand heated for a period of time (e.g., 83 C for 3 minutes). The lysateplates, along with Substrate half deep blocks, reaction plates (e.g.,384 well Griener Plates) and tips are moved from the Carousel or theheat block to the BIOMEK and test substrate (e.g., 10 ul) is dispensedinto each well of the reaction plate. An aliquot of lysate (e.g., 5 ul)is transferred from each well of a lysate plate to a corresponding wellof the reaction plate. Each well is overlaid with mineral oil (e.g., 6.5ul). The reaction plate is moved by the robot arm to the incubator andincubated (e.g., at 63 C) for one hour. The plate is then moved to thefluorescence plate reader, fluorescence is measured for each well, andthe robot arm returns the completed assay plate all other plate and tipcomponents to the carousel. These steps are repeated until all desiredassays are completed. When a primary screen indicates a desired changein activity, the clones cultured to produce additional enzyme (e.g., ininducing conditions), the enzyme may be purified and used both to verifythe initial result and for use in further characterization. The clonesexpressing the enzymes of interest may also be sequenced to identify orverify the mutations. In certain embodiments, 48 plates of cultures aregrown each day. In some embodiments, 12 to 16 lysis plates are processedthrough detection assays in an 8 to 10 hour day. In other embodiments,18 to 21 lysis plates are processed through detection assays in a 12 to15 hour day. Further details of embodiments of this system are providedin Example 67.

IX. FEN Endonuclease-Substrate Complexes

Structure-specific 5′ nucleases have been isolated from differentorganisms including bacteriophages (Hollingsworth & Nossal, 1991; Sayers& Eckstein, 1990), eubacteria (Deutscher & Kornberg, 1969; Klenow &Overgaard-Hansen, 1970; Lyamichev et al., 1993), archaea (Hosfield etal., 1998a; Hwang et al., 1998; Kaiser et al., 1999), yeast (Habraken etal., 1993; Harrington & Lieber, 1994b) and mammals (Harrington & Lieber,1994a; Lindahl et al., 1969; Murante et al., 1994; O'Donovan et al.,1994b). The ubiquitous presence of these enzymes is explained by theiressential role in Okazaki fragment processing (Goulian et al., 1990;Lundquist & Olivera, 1982; Turchi & Bambara, 1993) and repair of DNAdamage caused by alkylating agents or UV radiation (Habraken et al.,1993; Murray et al., 1994; O'Donovan et al., 1994a). In Okazaki fragmentprocessing, displacement synthesis by DNA polymerases generates branchedDNA structures in which the upstream and downstream strands overlap andcompete for the same sequence of the template strand. The branchedstructure can exist in multiple conformations depending on the positionof the branch point between the upstream and downstream strands on theshared template sequence (Lundquist & Olivera, 1982; Reynaldo et al.,2000). The structure-specific 5′ nucleases known as flap endonucleases(FEN1) (Harrington & Lieber, 1994a) specifically recognize aconformation called the overlap flap or 3′ one-nucleotide double-flapstructure in which the upstream strand, excluding the 3′ end nucleotide,is annealed to the template strand, displacing the 5′ portion of thedownstream strand (Kaiser et al., 1999; Kao et al., 2002; Lyamichev etal., 1999). FEN1 cleaves the downstream strand of the overlap flapstructure precisely after the first base-paired nucleotide, creating aligatable nick (Kaiser et al., 1999; Kao et al., 2002).

The conclusion that the 3′ end nucleotide of the upstream strand is notbase-paired with the template has been put forth from the observationthat any of the four natural bases at this position can supportefficient cleavage (Lyamichev et al., 1999). It was suggested that the3′ end nucleotide of the upstream strand interacts with the enzyme toposition the substrate in an optimal orientation for cleavage. Thedemonstration that sugar modifications of the 3′ end nucleotide inhibitactivity of FEN1 enzymes has further supported this hypothesis (Kaiseret al., 1999; Kao et al., 2002). The importance of the overlapping 3′end nucleotide of the upstream strand was not originally recognized, andmany laboratories characterized FEN1 enzymes using a flap structurewhich included adjacent upstream and downstream strands annealed to atemplate, but lacking a gap or overlap (Harrington & Lieber, 1994b).FEN1 enzymes cleave such a flap substrate inefficiently producing amajority of products that are not ligatable. The existence of theseproducts can be explained by the formation of alternative structureswith bulged nucleotides stabilized by the 5′ nuclease in an effort toforce the overlap flap structure (Kaiser et al., 1999; Kao et al., 2002;Lyamichev et al., 1999).

X-ray crystal structures have been determined for six structure-specific5′ nucleases: the archaeal FEN1 enzymes from Pyrococcus furiosus(PfuFEN1) (Hosfield et al., 1998b), Methanococcus jannaschii (MjaFEN1)(Hwang et al., 1998) and Pyrococcus horikoshii (PhoFEN1) (Matsui et al.,2002); the 5′ nuclease domain of eubacterial DNA polymerase from Thermusaquaticus (TaqExo) (Kim et al., 1995); the 5′-3′ exonuclease frombacteriophage T5 (Ceska et al., 1996), and the RNase H enzyme frombacteriophage T4 (Mueser et al., 1996). These structures reveal a commona/b topology and similar structural motifs despite a low amino acidsequence identity and similarity between the archaeal, eubacterial andbacteriophage FEN1 groups (see, for example, (Hosfield et al., 1998b)).All of these enzymes have been shown to bind two divalent metal ionswhich form a complex network of interactions with highly conservedacidic amino acids lying at the bottom of a positively charged cleft.One metal ion is presumably involved in catalysis and the other in theDNA binding (Shen et al., 1996). In the PfuFEN1 structure, the magnesiumion M-1, involved in catalysis, is located in close proximity to thecluster of amino acids Asp27, Asp80, Glu152, and Glu154; and themagnesium ion M-2 involved in substrate binding interacts with thecluster of amino acids Asp173, Asp175, and Asp236 approximately 5 Å fromM-1.

The helical arch is a common structural motif shared by the FEN1 enzymesand was originally identified in the T5 5′-3′ exonuclease structure(Ceska et al., 1996). The arch is located close to the enzyme's activesite and forms a flexible loop that can accommodate single-stranded butnot double-stranded DNA. The motif provides structural support for thehypothesis that the 5′ flap of DNA substrate threads through a hole totranslocate DNA to the enzyme's active site. The threading mechanism wasoriginally proposed to explain biochemical data that blocking the free5′ end of the flap with a bulky modification or rendering itdouble-stranded using a complementary oligonucleotide suppresses thecleavage efficiency and can even trap FEN1 on the 5′ flap (Lyamichev etal., 1993; Murante et al., 1995). While most studies agree on thethreading mechanism, Bambara and his group have shown that a variety ofbulky flap modifications can be tolerated by human FEN1 endonuclease(Bornarth et al., 1999).

Another common fold shared by the FEN1 enzymes is thehelix-hairpin-helix (HhH) motif found in many enzyme families (Dohertyet al., 1996; Shao & Grishin, 2000). This type of fold is involved innon-sequence-specific binding of duplex DNA via interactions with thesugar-phosphate backbone of one of the strands (Pelletier et al., 1996;Thayer et al., 1995). Together with the helical arch and network ofamino acids interacting with the M-1 and M-2 ions, the HhH motif definesa positively charged active-site DNA-binding groove in FEN1. In PfuFEN1,the DNA-binding groove is 32 Å wide and 44 Å long, suggesting that itcan accommodate a 12 base-pair double-stranded DNA (Hosfield et al.,1998b). Biochemical analysis of point mutations at the DNA-bindinggroove of the FEN1 enzymes revealed conserved amino acids on the surfaceof the groove involved in catalysis and substrate binding (Bhagwat etal., 1997; Garforth et al., 1999; Matsui et al., 2002; Qiu et al., 2002;Shen et al., 1996; Shen et al., 1997; Xu et al., 1997).

Structural and functional similarity between the 5′ nucleases suggests acommon mechanism for substrate binding and catalysis for all enzymes inthis family. In the absence of co-crystal or NMR structures for a 5′nuclease/DNA complex, several models of the complex have been proposedto elucidate the mechanism of substrate binding (Ceska et al., 1996;Dervan et al., 2002; Hosfield et al., 1998b; Hwang et al., 1998). Thesemodels suggest that the substrate binds at the active-site DNA-bindinggroove with the cleavable phosphodiester linkage close to the metal ioninvolved in catalysis and with the 5′ flap threading through the helicalarch.

Methylphosphonate and 2′-O-methyl substitutions have proven to bepowerful methods for identifying contacts between nucleic acids andproteins (Botfield & Weiss, 1994; Dertinger & Uhlenbeck, 2001; Hou etal., 2001; Noble et al., 1984; Pritchard et al., 1994; Smith &McLaughlin, 1997). Methylphosphonate substitutions are almost isostericwith phosphodiester linkages but unlike phosphodiester linkages areneutral and therefore can be used to identify ionic interactions inprotein/substrate complexes without introducing steric clashes with theproteins (Dertinger & Uhlenbeck, 2001). Methylphosphonate linkages havebeen shown to induce local bending in the double helical DNA axis by themechanism of asymmetric phosphate charge neutralization. However, thebending angle estimated as 3.5o per methylphosphonate substitution(Tomky et al., 1998) is comparable to the intrinsic sequence-specificDNA bending (Goodsell et al., 1993) and thermal flexibility of duplexDNA of ˜7o per base pair estimated from its persistence length (Cantor &Schimmel, 1980). Substitution of a methyl group in place of anon-bridging oxygen in the phosphodiester linkage at a point ofelectrostatic contact with a protein usually decreases the affinity ofsubstrate binding (Dertinger & Uhlenbeck, 2001). This property ofmethylphosphonate modifications makes unnecessary, in most cases, theseparation of Rp and Sp stereoisomers of chemically introducedmethylphosphonate linkages and justifies the use of their racemicmixtures. 2′-O-methyl substitutions replace the 2′ proton in thedeoxyribose ring with a bulky O-methyl group with two major outcomes forduplex DNA structure. First, 2′-O-methyl groups change theconformational preference of ribose from C2′-endo to C3′-endo sugarpuckering, forcing a local transition from B-form to A-form DNA. Second,they introduce steric clashes at sites of contacts with the proteins(Hou et al., 2001). In this work, we introduced a singlemethylphosphonate substitution into each phosphodiester linkage of theoverlap flap DNA substrate to map phosphates interacting with PfuFEN1.Similarly, we introduced 2′-O-methyl substitutions to identify stericcontacts in the PfuFEN1/DNA complex. Using the three-dimensionalstructure of PfuFEN1 (Hosfield et al., 1998b) and a modeled structure ofthe overlap flap substrate, we performed energy minimization andmolecular dynamics (MD) simulations to test two alternative structuresof the PfuFEN1/DNA complex. The model consistent with themethylphosphonate data was used to identify candidate amino acidscontacting phosphates in the substrate. To confirm the predictedinteractions, PfuFEN1 variants mutated at these amino acids were testedon the methylphosphonate substrates. The confirmed interactions wereused as restraints in MD simulations to develop a detailed model of thePfuFEN1/DNA complex.

X. The INVADER Assay for Direct Detection and Measurement of SpecificAnalytes.

The following description provides illustrative examples of targetsequence detection through the use of the compositions and methods ofthe present invention. These example include the detection of humancytomegalovirus viral DNA, single nucleotide polymorphisms in the humanapolipoprotein E gene, mutations in the human hemochromatosis gene,mutations in the human MTHFR, prothrombin 20210GA polymorphism, the HR-2mutation in the human Factor V gene, single nucleotide polymorphisms inthe human TNF-α Gene, and Leiden mutation in the human Factor V gene.Included in these descriptions are novel nucleic acid compositions foruse in the detection of such sequence. Examples 54-61 below providedetails on the design and execution of these illustrative embodiments.It is understood that these detection assays may be performed alone,e.g., in individual detection assays, or they may be performed incombinations. Combinations may comprise multiplex analyses, e.g.,wherein a plurality of different target sequences are detected in asingle reaction (e.g., by using a different quenched dye on a FRET probefor each sequence suspected to be present in a sample or mixture).Combinations may comprise panels, wherein a plurality of detectionreactions are performed simultaneously, e.g., on an assay plate.

A. Detection of Human Cytomegalovirus Viral DNA by Invasive Cleavage

Human cytomegalovirus (HCMV) causes, or is associated with, a widevariety of diseases in humans (Table 3). More than 90% of bone marrow orkidney transplant recipients (immunocompromised hosts) develop HCMVinfections, most of which are due to reactivation of latent virus byimmunosuppressive drugs, as well as transmission of virus by latentlyinfected donor tissue or blood (Ackerman et al., Transplant. Proc.,20(S1):468 [1988]; and Peterson et al., Medicine 59:283 [1980]).

TABLE 3 Disease Caused By Human Cytomegalovirus cytomegalic inclusionheterophil-negative disease in neonates mononucleosis interstitialpneumonia pneumonitis retinitis hepatitis pancreatitismeningoencephalitis gastrointestinal disease disseminated infection

There are instances in which rapid, sensitive, and specific diagnosis ofHCMV disease is imperative. In recent years, the number of patientsundergoing organ and tissue transplantations has increased markedly.HCMV is the most frequent cause of death in immunocompromised transplantrecipients, thereby confirming the need for rapid and reliablelaboratory diagnosis. Lymphocytes, monocytes, and possibly arterialendothelial or smooth muscle cells, are sites of HCMV latency.Therefore, prevention of HCMV infections in immunocompromisedindividuals (e.g., transplant recipients) includes use of HCMV-negativeblood products and organs. Additionally, HCMV can be spreadtransplacentally, and to newborns by contact with infected cervicalsecretions during birth. Thus, a rapid, sensitive, and specific assayfor detecting HCMV in body fluids or secretions may be desirable as ameans to monitor infection, and consequently, determine the necessity ofcesarean section.

Diagnosis of HCMV infection may be performed by conventional cellculture using human fibroblasts; shell vial centrifugation cultureutilizing monoclonal antibodies and immunofluorescent stainingtechniques; serological methods; the HCMV antigenemia assay whichemploys a monoclonal antibody to detect HCMV antigen in peripheral bloodleukocytes (PBLs); or by nucleic acid hybridization assays. Thesevarious methods have their advantages and limitations. Conventional cellculture is sensitive but slow, as cytopathic effect (CPE) may take 30 ormore days to develop. Shell vial centrifugation is more rapid but stillrequires 24-48 hours for initial results. Both culture methods areaffected by antiviral therapy. In immunocompromised patients, theability to mount IgG and/or IgM antibody responses to HCMV infection areimpaired, and serological methods are thus not reliable in this setting.Alternatively, IgM antibodies may be persistent for months afterinfection is resolved, and thus their presence may not be indicative ofactive infection. The HCMV antigenemia assay is labor intensive and isnot applicable to specimens other than PBLs.

Recent advances in molecular biology have spurred the use of DNA probesin attempts to provide a more rapid, sensitive and specific assay fordetecting HCMV in clinical specimens. For example, radiolabeled DNAprobes have been used to hybridize to tissue cultures infected with orby HCMV, or in clinical samples suspected of containing HCMV(“hybridization assays”). However, probing of tissue cultures requiresat least 18-24 hours for growth to amplify the antigen (HCMV) to bedetected, if present, and additional time for development ofautoradiographic detection systems. Using hybridization assays forassaying clinical specimens for HCMV may lack sensitivity, dependingupon the titer of virus and the clinical sample assayed. Detection ofHCMV in clinical samples has been reported using the polymerase chainreaction (PCR) to enzymatically amplify HCMV DNA. Methods using PCRcompare favorably with virus isolation, in situ hybridization assays,and Southern blotting; See, e.g., Bamborschke et al,. J. Neurol.,239:205 [1992]; Drouet et al., J. Virol. Meth., 45:259 [1993]; Einseleet al., Blood 77:1104-1110 [1991]; Einsele et al., Lancet 338:1170[1991]; Lee et al., Aust. NZ J. Med., 22:249 [1992]; Miller et al., J.Clin. Microbiol., 32:5 [1994]; Rowley et al., Transplant. 51:1028[1991]; Spector et al. J. Clin. Microbiol., 30:2359 [1992]; and Stanieret al., Mol. Cell. Probes 8:51 [1992]). Others, comparing the HCMVantigenemia assay with PCR methods, have found PCR methods as efficientor slightly more efficient in the detection of HCMV (van Dorp et al.(1992) Transplant. 54:661; Gerna et al. (1991) J. Infect. Dis. 164:488;Vleiger et al. (1992) Bone Marrow Transplant. 9:247; Zipeto et al.(1992) J. Clin. Microbiol. 30:527]. In addition, PCR methods haveexhibited great sensitivity when specimens other than PBLs are assayed(Natori et al., Kansenshogaku Zasshi 67:1011 [1993]; Peterson et al.,Medicine 59:283 [1980]; Prosch et al., J. Med. Virol., 38:246 [1992];Ratnamohan et al., J. Med. Virol. 38:252 [1992]). However, because ofthe dangers of false positive reactions, these PCR-based proceduresrequire rigid controls to prevent contamination and carry over (Ehrlichet al., in PCR-Based Diagnostics in Infectious Diseases, Ehrlich andGreenberg (eds), Blackwell Scientific Publications, [1994], pp.3-18).Therefore, there exists a need for a rapid, sensitive, and specificassay for HCMV that has a reduced risk of false positive result due tocontamination by reaction product carried over from other samples.

As shown herein, the INVADER-directed cleavage assay is rapid, sensitiveand specific. Because the accumulated products do not contribute to thefurther accumulation of signal, reaction products carried over from onestandard (i.e., non-sequential) INVADER-directed cleavage assay toanother cannot promote false positive results. The use of multiplesequential INVADER-directed cleavage assays will further boost thesensitivity of HCMV detection without sacrifice of these advantages.

B. Detection of Single Nucleotide Polymorphisms in the HumanApolipoprotein E Gene

Apolipoprotein E (ApoE) performs various functions as a proteinconstituent of plasma lipoproteins, including its role in cholesterolmetabolism. It was first identified as a constituent ofliver-synthesized very low density lipoproteins which function in thetransport of triglycerides from the liver to peripheral tissues. Thereare three major isoforms of ApoE, referred to as ApoE2, ApoE3 and ApoE4which are products of three alleles at a single gene locus. Threehomozygous phenotypes (Apo-E2/2, E3/3, and E4/4) and three heterozygousphenotypes (ApoE3/2, E4/3 and E4/2) arise from the expression of any twoof the three alleles. The most common phenotype is ApoE3/3 and the mostcommon allele is E3. See Mahley, R. W., Science 240:622-630 (1988).

The amino acid sequences of the three types differ only slightly. ApoE4differs from ApoE3 in that in ApoE4 arginine is substituted for thenormally occurring cysteine at amino acid residue 112. The most commonform of ApoE2 differs from ApoE3 at residue 158, where cysteine issubstituted for the normally occurring arginine. See Mahley, Science,supra.

The frequency of the apoE4 allele has been shown to be markedlyincreased in sporadic Alzheimer's Disease (AD) (Poirier, J. et al.,1993, Apolipoprotein E phenotype and Alzheimer's Disease, Lancet,342:697-699; Noguchi, S. et al., 1993, Lancet (letter), 342:737) andlate onset familial Alzheimer's disease (AD) (Corder, E. H. et al.,1993, Science, 261:921-923; Payami, H. et al., 1993, Lancet (letter),342:738). This gene dosage effect was observed in both sporadic andfamilial cases (i.e., as age of onset increases, E4 allele copy numberdecreases). Women, who are generally at a greater risk of developingAlzheimer's disease, show increased E4 allele frequency when compared toage matched men.

C. Detection of Mutations in the Human Hemochromatosis Gene

Hereditary hemochromatosis (HH) is an inherited disorder of ironmetabolism wherein the body accumulates excess iron. In symptomaticindividuals, this excess iron leads to deleterious effects by beingdeposited in a variety of organs leading to their failure, and resultingin cirrhosis, diabetes, sterility, and other serious illnesses.

HH is inherited as a recessive trait; heterozygotes are asymptomatic andonly homozygotes are affected by the disease. It is estimated thatapproximately 10% of individuals of Western European descent carry an HHgene mutation and that there are about one million homozygotes in theUnited States. Although ultimately HH produces debilitating symptoms,the majority of homozygotes have not been diagnosed. Indeed, it has beenestimated that no more than 10,000 people in the United States have beendiagnosed with this condition. The symptoms are often confused withthose of other conditions, and the severe effects of the disease oftendo not appear immediately. It would be desirable to provide a method toidentify persons who are ultimately destined to become symptomatic inorder to intervene in time to prevent excessive tissue damage. Onereason for the lack of early diagnosis is the inadequacy of presentlyavailable diagnostic methods to ascertain which individuals are at risk.

Although blood iron parameters can be used as a screening tool, aconfirmed diagnosis often employs HLA typing, which is tedious,nonspecific, and expensive and/or liver biopsy which is undesirablyinvasive and costly. Accordingly, others have attempted to developinexpensive and noninvasive diagnostics both for detection ofhomozygotes having existing disease, in that presymptomatic detectionwould guide intervention to prevent organ damage, and for identificationof carriers. The need for such diagnostics is documented for example, inFinch, C. A. West J Med (1990) 153:323-325; McCusick, V. et al.Mendelian Inheritance in Man 11th ed., Johns Hopkins University Press(Baltimore, 1994) pp. 1882-1887; Report of the Joint World HealthOrganization/HH Foundation/French HH Association Meeting, 1993.

D. Detection of Mutations in the Human MTHFR

Folic acid derivatives are coenzymes for several critical single-carbontransfer reactions, including reactions in the biosynthesis of purines,thymidylate and methionine. Methylenetetrahydrofolate reductase (MTHFR;EC 1.5.1.20) catalyzes the NADPH-linked reduction of5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, aco-substrate for methylation of homocysteine to methionine. The porcineliver enzyme, a flavoprotein, has been purified to homogeneity; it is ahomodimer of 77-kDa subunits. Partial proteolysis of the porcine peptidehas revealed two spatially distinct domains: an N-terminal domain of 40kDa and a C-terminal domain of 37 kDa. The latter domain contains thebinding site for the allosteric regulator S-adenosylmethionine.

Hereditary deficiency of MTHFR, an autosomal recessive disorder, is themost common inborn error of folic acid metabolism. A block in theproduction of methyltetrahydrofolate leads to elevated homocysteine withlow to normal levels of methionine. Patients with severe deficiencies ofMTHFR (0-20% activity in fibroblasts) can have variable phenotypes.Developmental delay, mental retardation, motor and gait abnormalities,peripheral neuropathy, seizures and psychiatric disturbances have beenreported in this group, although at least one patient with severe MTHFRdeficiency was asymptomatic. Pathologic changes in the severe forminclude the vascular changes that have been found in other conditionswith elevated homocysteine, as well as reduced neurotransmitter andmethionine levels in the CNS. A milder deficiency of MTHFR (35-50%activity) has been described in patients with coronary artery disease.Genetic heterogeneity is likely, considering the diverse clinicalfeatures, the variable levels of enzyme activity, and the differentialheat inactivation profiles of the reductase in patients' cells. Methodsto detect the MTHFR mutation include: AS-PCR (Hessner, et al. Br JHaematol 106, 237-9 (1999)) and PCR-RFLP (Nature Genetics, Frosst etal.1995:10; 111-113).

E. Detection of Prothrombin 20210GA Polymorphism and the Factor V LeidenPolymorphism

The coagulation cascade is a complex series of zymogen activations,inactivations and feed back loops involving numerous enzymes and theircofactors. The entire cascade, from tissue injury or venous trauma toclotting has been well described (refs). The cascade culminates in theconversion of prothrombin (Factor II) to thrombin. This is catalyzed bythe activated form of factor X, factor Xa and its cofactor, activatedfactor V, factor Va. Thrombin then converts fibrinogen to fibrin andpromotes fibrin cross-linking and clot formation by activating factorXIII. In addition to the above stated functions, thrombin a serineprotease, can also activate factor V in a positive feed-back loop.Factor Va is a pro-coagulant cofactor in the clotting cascade, and whenclot formation is sufficient, is inactivated by activated protein C(APC).

Venous thrombosis is the obstruction of the circulation by clots thathave been formed in the veins or have been released from a thrombusformed elsewhere. The most frequent sites of clot formation are the deepveins of the legs, but it also may occur in veins in the brain, retina,liver and mesentery. Factors other than heritable defects that can playa role in the development of thrombosis include recent surgery,malignant disorders, pregnancy and labor and long term immobilization.

Studies of hereditary thrombophilia, defined as an increased tendencytowards venous thrombotic disease in relatively young adults, haveprovided insights into the genetic factors that regulate thrombosis. In1993, Dahlback et al. (Proc Natl Acad Sci USA 1993;90:1004-1008)described an insensitivity to APC, a critical anti-coagulant in theclotting cascade, in three unrelated families with hereditarythrombophilia. The anticoagulant property of APC resides in its capacityto inactivate the activated cofactors Va and VIII by limited proteolysis(ref 3). This inactivation of cofactors Va and VIIIa results inreduction of the rate of formation of thrombin, the end product of thecascade. This observation was confirmed by other investigators (ref) andthe term “APC resistance” was coined to describe this particularphenotype in thrombophilic patients. In a subsequent study of 20families with thrombophilia and APC resistance, an autosomal dominantpattern of inheritance was observed (17). Bertina et al (Nature, 1994,May 5;369 (6475):64-7) then demonstrated that the phenotype of APCresistance is associated with heterozygosity or homozygosity for asingle point mutation at nucleotide 1691 in exon 10 of the factor Vgene. This single base change, a guanine to adenine substitution, yieldsa mutant factor V molecule wherein the arginine at position 506 isreplaced with glutamine. This form of the factor V molecule,characterized at Leiden University, (Bertenia et al) is known as the FVQ506 or FV Leiden mutation, and is inactivated less efficiently by APCthan the wild type protein. It has been postulated that the prolongedcirculation of activated factor V promotes a hypercoagulable state andincreases the risk of thrombosis. Subsequent analysis of various patientgroups exhibiting symptoms of venous thrombosis indicate that the factorV Leiden mutation is the single most common heritable factorcontributing to an increased risk of venous thrombosis.

In 1996, studies by Poort et al. (Blood. 1996:88; 3698-703) revealed thesecond most common heritable factor contributing to increase thromboticrisk. In studying the sequence of the prothrombin gene in selectedpatients with a documented familial history of venous thrombophilia, thePoort group identified a single point mutation in the 3′ untranslatedregion. This G to A transition at position 20210 is strongly correlatedwith elevated plasma prothrombin levels, and was also shown to beassociated with an almost threefold increased risk of venous thrombosis(abstract, Howard)

The first reported case of a thrombophilia pateint geneticallyhomozygous for the G to A polymorphism in the 3′ untranslated region wasby Howard, et al (Blood Coagulation Fibrinolysis 1997 Jul;8(5):316-9).The patient, a healthy young Mexican male presented with a myocardialinfarction, venous thrombosis and embolism. The patient was found to behomozygous for the prothrombin mutation and heterozygous for the FactorV Leiden mutation, supporting the doublehit theory for thrombophilia inyoung patients.

Studies by Hessner et al. show that the prothrombin 20210GA genotype wasnearly 5 times as prevalent in the symptomoatic FVL carriers than in arandom Caucasian control group (British Journal of Haematology, 1999,106), and that allele frequencies for the prothrombin and Factor Vmutants vary among different ethnic backgrounds (Thromb Haemostat 1999;81:733-8). The above discussion confirms that early detection of thefactor V Leiden mutation and the factor II prothrombin mutation areparamount in hereditary thrombotic risk assessment. The nature of thesetwo mutations, that is, a single base change in the nucleic acidsequence, make them amenable to a variety of nucleic acid detectionmethods known to the art, though the demand for faster, more reliable,cost-effective and user-friendly tests for the detection of specificnucleic acid sequences continues to grow. The most common methods totest for these mutations include PCR/RFLP, AS-PCR and functional,coagulation assay.

F. Detection of the HR-2 Mutation in the Human Factor V Gene

The R-2 polymorphism is located in exon 13 of the factor V gene, and isthe result of an A to G transition at base 4070, replacing the wild-typeamino acid histine with the mutant argenine in the mature protein. TheR-2 polymorphism is one of a set of mutations termed collectively HR-2.The HR-2 haplotype is defined by 6 nucleotide base substitutions inexons 13 and 16 of the factor V gene. The haplotype is associated withan increased functional resistance to activated protein C both in normalsubjects and in thrombophilic patients. When present as a compoundheterozygote in conjunction with the factor V Leiden mutation, clinicalsymptoms are comparable to those seen in patients homozygous for thefactor V Leiden mutation, and include increased risk of deep veinthrombosis.

G. Detection of Single Nucleotide Polymorphisms in the Human TNF-αGene

The human cytokine tumor necrosis factor alpha (TNF-alpha) has beenshown to be a major factor in graft rejection; the more TNF-alphapresent in the system, the greater the rejection response totransplanted tissue. Mutations in TNF-alpha have also been correlatedwith cerebral malaria (Nature 1994;371:508-510), fulminas purpura (JInfect Dis. 1996;174:878-880), and mucocutaneous leishmaniaisis (J ExpMed. 1995;182:1259-1264). The mutation detected in this example islocated in the promoter region of the TNF-alpha gene at position minus308. The wild-type guanine (G) is replaced with a mutant adenine (A).This result of this promoter mutation is the enhancement oftranscription of TNF-alpha by 6-7 fold. Methods to detect mutations inTNF-alpha include sequencing, denaturing gradient gel electorphoresis,PCR methods, and methods involving both PCR and post-PCR hybridizationwith specific oligos.

H. Detection of Methicillin Resistant Staphylococcus aureus

Staphylococcus aureus is recognized as one of the major causes ofinfections in humans occurring in both in the hospital and in thecommunity at large. One of the most serious concerns in treating anybacterial infection is the increasing resistance to antibiotics. Thegrowing incidence of methicillin-resistant S. aureus (MRSA) infectionsworldwide has underscored the importance of both early detection of theinfective agent, and defining a resistance profile such that propertreatment can be given. The primary mechanism for resistance tomethicillin involves the production of a protein called PBP2a, encodedby the mecA gene. The mecA gene not specific to Staphalococcus aureus,but is of extraspecies origin. The mecA gene is however, indicative ofmethicillin resistance and is used as a marker for the detection ofresistant bacteria. So, to identify methicillin resistant S. aureus vianucleic acid techniques, both the mecA gene and at least one speciesspecific gene must be targeted. A particular species specific gene, thenuclease or nuc gene is used in the following example. Methods used todetect MRSA include time consuming and laborious culturing andcoagulation assays and growth assays on antibiotic media. Molecularapproaches include a Cycling Probe™ assay, the Velogene™ Kit fromAlexon-Trend (Ramsey, MN cat # 818-48), anti-body test which bind thePBP2a protein, bDNA Assay (Chiron, Emeryville, Calif.), all of whichtests only for the presence of the mecA gene and are not Staph. aureusspecific.

XI. Kits

In some embodiments, the present invention provides kits comprising oneor more of the components necessary for practicing the presentinvention. For example, the present invention provides kits for storingor delivering the enzymes of the present invention and/or the reactioncomponents necessary to practice a cleavage assay (e.g., the INVADERassay). The kit may include any and all components necessary or desiredfor the enzymes or assays including, but not limited to, the reagentsthemselves, buffers, control reagents (e.g., tissue samples, positiveand negative control target oligonucleotides, etc.), solid supports,labels, written and/or pictorial instructions and product information,inhibitors, labeling and/or detection reagents, package environmentalcontrols (e.g., ice, desiccants, etc.), and the like. In someembodiments, the kits provide a sub-set of the required components,wherein it is expected that the user will supply the remainingcomponents. In some embodiments, the kits comprise two or more separatecontainers wherein each container houses a subset of the components tobe delivered. For example, a first container (e.g., box) may contain anenzyme (e.g., structure specific cleavage enzyme in a suitable storagebuffer and container), while a second box may contain oligonucleotides(e.g., INVADER oligonucleotides, probe oligonucleotides, control targetoligonucleotides, etc.).

Additionally, in some embodiments, the present invention providesmethods of delivering kits or reagents to customers for use in themethods of the present invention. The methods of the present inventionare not limited to a particular group of customers. Indeed, the methodsof the present invention find use in the providing of kits or reagentsto customers in many sectors of the biological and medical community,including, but not limited to customers in academic research labs,customers in the biotechnology and medical industries, and customers ingovernmental labs. The methods of the present invention provide for allaspects of providing the kits or reagents to the customers, including,but not limited to, marketing, sales, delivery, and technical support.

In some embodiments of the present invention, quality control (QC)and/or quality assurance (QA) experiments are conducted prior todelivery of the kits or reagents to customers. Such QC and QA techniquestypically involve testing the reagents in experiments similar to theintended commercial uses (e.g., using assays similar to those describedherein). Testing may include experiments to determine shelf life ofproducts and their ability to withstand a wide range of solution and/orreaction conditions (e.g., temperature, pH, light, etc.).

In some embodiments of the present invention, the compositions and/ormethods of the present invention are disclosed and/or demonstrated tocustomers prior to sale (e.g., through printed or web-based advertising,demonstrations, etc.) indicating the use or functionality of the presentinvention or components of the present invention. However, in someembodiments, customers are not informed of the presence or use of one ormore components in the product being sold. In such embodiments, salesare developed, for example, through the improved and/or desired functionof the product (e.g., kit) rather than through knowledge of why or howit works (i.e., the user need not know the components of kits orreaction mixtures). Thus, the present invention contemplates makingkits, reagents, or assays available to users, whether or not the userhas knowledge of the components or workings of the system.

Accordingly, in some embodiments, sales and marketing efforts presentinformation about the novel and/or improved properties of the methodsand compositions of the present invention. In other embodiments, suchmechanistic information is withheld from marketing materials. In someembodiments, customers are surveyed to obtain information about the typeof assay components or delivery systems that most suits their needs.Such information is useful in the design of the components of the kitand the design of marketing efforts.

EXAMPLES

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the disclosure which follows, the following abbreviations apply: Afu(Archaeoglobus fulgidus); Mth (Methanobacterium thermoautotrophicum);Mja (Methanococcus jannaschii); Pfu (Pyrococcus furiosus); Sso(Sulfolobus solfataricus); Pae (Pyrobaculum aerophilumI); Tli(Thermococcus litoralis); Ave (Archaeaglobus veneficus); Apr(Archaeaglobus profundus); Abr (Acidianus brierlyi); Aam (Acidianusambivalens); Dam (Desulfurococcus amylolyticus); Dmo (Desulfurococcusmobilis); Pbr (Pyrodictium brockii); Tgo (Thermococcus gorgonarius); Tzi(Thermococcus zilligii); Mke (Methanopyrus kandleri); Mig (Methanococcusigneus); Pho (Pyrococcus horikoshii); Ape (Aeropyrum pernix); Pwo(Pyrococcus woesei); Taq (Thermus aquaticus); Taq DNAP, DNAPTaq, and TaqPol I (T. aquaticus DNA polymerase I); DNAPStf (the Stoffel fragment ofDNAPTaq); DNAPEc1 (E. coli DNA polymerase I); Tth (Thermusthermophilus); Ex. (Example); Fig. (Figure); ° C. (degrees Centigrade);g (gravitational field); hr (hour); min (minute); olio(oligonucleotide); rxn (reaction); vol (volume); w/v (weight to volume);v/v (volume to volume); BSA (bovine serum albumin); CTAB(cetyltrimethylammonium bromide); HPLC (high pressure liquidchromatography); DNA (deoxyribonucleic acid); p (plasmid); μl(microliters); ml (milliliters); μg (micrograms); mg (milligrams); M(molar); mM (milliMolar); μM (microMolar); pmoles (picomoles); amoles(attomoles); zmoles (zeptomoles); nm (nanometers); kdal (kilodaltons);OD (optical density); EDTA (ethylene diamine tetra-acetic acid); FITC(fluorescein isothiocyanate); SDS (sodium dodecyl sulfate); NaPO₄(sodium phosphate); NP-40 (Nonidet P-40); Tris(tris(hydroxymethyl)-aminomethane); PMSF (phenylmethylsulfonylfluoride);TBE (Tris-Borate-EDTA, i.e., Tris buffer titrated with boric acid ratherthan HCl and containing EDTA); PBS (phosphate buffered saline); PPBS(phosphate buffered saline containing 1 mM PMSF); PAGE (polyacrylamidegel electrophoresis); Tween (polyoxyethylene-sorbitan); ATCC (AmericanType Culture Collection, Rockville, Md.); Coriell (Coriell CellRepositories, Camden, N.J.); DSMZ (Deutsche Sammlung von Mikroorganismenund Zellculturen, Braunschweig, Germany); Red dye (REDMOND RED dye,Synthetic Genetics, San Diego, Calif.); Z28 (ECLIPSE Quencher, SyntheticGenetics, San Diego, Calif.); Ambion (Ambion, Inc., Austin, Tex.);Boehringer (Boehringer Mannheim Biochemical, Indianapolis, Ind.); MJResearch (MJ Research, Watertown, Mass.; Sigma (Sigma Chemical Company,St. Louis, Mo.); Dynal (Dynal A.S., Oslo, Norway); Gull (GullLaboratories, Salt Lake City, Utah); Epicentre (Epicentre Technologies,Madison, Wis.); Lampire (Biological Labs., Inc., Coopersberg, Pa.); MJResearch (MJ Research, Watertown,Mass.); National Biosciences (NationalBiosciences, Plymouth, Minn.); NEB (New England Biolabs, Beverly,Mass.); Novagen (Novagen, Inc., Madison, Wis.); Promega (Promega, Corp.,Madison, Wis.); Stratagene (Stratagene Cloning Systems, La Jolla,Calif.); Clonetech (Clonetech, Palo Alto, Calif.) Pharmacia (Pharmacia,Piscataway, N.J.); Milton Roy (Milton Roy, Rochester, N.Y.); Amersham(Amersham International, Chicago, Ill.); and USB (U.S. Biochemical,Cleveland, Ohio). Glen Research (Glen Research, Sterling, Va.); Coriell(Coriell Cell Repositories, Camden, N.J.); Gentra (Gentra, Minneapolis,Minn.); Third Wave Technologies (Third Wave Technologies, Madison,Wis.); PerSeptive Biosystems (PerSeptive Biosystems, Framington, Mass.);Microsoft (Microsoft, Redmond, Wash.); Qiagen (Qiagen, Valencia,Calif.); Molecular Probes (Molecular Probes, Eugene, Oreg.); VWR (VWRScientific,); Advanced Biotechnologies (Advanced Biotechnologies, INC.,Columbia, Md.); Invitrogen (Invitrogen, Carlsbad, Calif.) and PerkinElmer (also known as PE Biosytems and Applied Biosystems, Foster City,Calif.).

Example 1 Characteristics of Native Thermostable DNA Polymerases

A. 5′ Nuclease Activity of DNAPTaq

During the polymerase chain reaction (PCR) (Saiki et al., Science239:487 [1988]; Mullis and Faloona, Meth. Enzymol., 155:335 [1987]),DNAPTaq is able to amplify many, but not all, DNA sequences. Onesequence that cannot be amplified using DNAPTaq is shown in FIG. 5(Hairpin structure is SEQ ID NO:15, FIG. 5 also shows a primer: SEQ IDNO:17) This DNA sequence has the distinguishing characteristic of beingable to fold on itself to form a hairpin with two single-stranded arms,which correspond to the primers used in PCR.

To test whether this failure to amplify is due to the 5′ nucleaseactivity of the enzyme, the abilities of DNAPTaq and DNAPStf to amplifythis DNA sequence during 30 cycles of PCR were compared. Syntheticoligonucleotides were obtained from The Biotechnology Center at theUniversity of Wisconsin-Madison. The DNAPTaq and DNAPStf were fromPerkin Elmer (i.e., AMPLITAQ DNA polymerase and the Stoffel fragment ofAMPLITAQ DNA polymerase). The substrate DNA comprised the hairpinstructure shown in FIG. 6 cloned in a double-stranded form into pUC19.The primers used in the amplification are listed as SEQ ID NOS:16-17.Primer SEQ ID NO:17 is shown annealed to the 3′ arm of the hairpinstructure in FIG. 5. Primer SEQ ID NO:16 is shown as the first 20nucleotides in bold on the 5′ arm of the hairpin in FIG. 5.

Polymerase chain reactions comprised 1 ng of supercoiled plasmid targetDNA, 5 pmoles of each primer, 40 μM each dNTP, and 2.5 units of DNAPTaqor DNAPStf, in a 50 μl solution of 10 mM Tris. Cl pH 8.3. The DNAPTaqreactions included 50 mM KCl and 1.5 mM MgCl₂. The temperature profilewas 95° C. for 30 sec., 55° C. for 1 min. and 72° C. for 1 min., through30 cycles. Ten percent of each reaction was analyzed by gelelectrophoresis through 6% polyacrylamide (cross-linked 29:1) in abuffer of 45 mM Tris·Borate, pH 8.3, 1.4 mM EDTA.

The results are shown in FIG. 6. The expected product was made byDNAPStf (indicated simply as “S”) but not by DNAPTaq (indicated as “T”).It was concluded that the 5′ nuclease activity of DNAPTaq is responsiblefor the lack of amplification of this DNA sequence.

To test whether the 5′ unpaired nucleotides in the substrate region ofthis structured DNA are removed by DNAPTaq, the fate of the end-labeled5′ arm during four cycles of PCR was compared using the same twopolymerases (FIG. 7). The hairpin templates, such as the one describedin FIG. 5, were made using DNAPStf and a ³²P-5′-end-labeled primer. The5′-end of the DNA was released as a few large fragments by DNAPTaq butnot by DNAPStf. The sizes of these fragments (based on their mobilities)show that they contain most or all of the unpaired 5′ arm of the DNA.Thus, cleavage occurs at or near the base of the bifurcated duplex.These released fragments terminate with 3′ OH groups, as evidenced bydirect sequence analysis, and the abilities of the fragments to beextended by terminal deoxynucleotidyl transferase.

FIGS. 8-10 show the results of experiments designed to characterize thecleavage reaction catalyzed by DNAPTaq. Unless otherwise specified, thecleavage reactions comprised 0.01 pmoles of heat-denatured, end-labeledhairpin DNA (with the unlabeled complementary strand also present), 1pmole primer (complementary to the 3′ arm) and 0.5 units of DNAPTaq(estimated to be 0.026 pmoles) in a total volume of 10 μl of 10 mMTris-Cl, ph 8.5, 50 mM KCl and 1.5 mM MgCl₂. As indicated, somereactions had different concentrations of KCl, and the precise times andtemperatures used in each experiment are indicated in the individualFigures. The reactions that included a primer used the one shown in FIG.5 (SEQ ID NO:17). In some instances, the primer was extended to thejunction site by providing polymerase and selected nucleotides.

Reactions were initiated at the final reaction temperature by theaddition of either the MgCl₂ or enzyme. Reactions were stopped at theirincubation temperatures by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. The T_(m) calculations listed were madeusing the Oligo™ primer analysis software from National Biosciences,Inc. These were determined using 0.25 μM as the DNA concentration, ateither 15 or 65 mM total salt (the 1.5 mM MgCl₂ in all reactions wasgiven the value of 15 mM salt for these calculations).

FIG. 8 is an autoradiogram containing the results of a set ofexperiments and conditions on the cleavage site. FIG. 8A is adetermination of reaction components that enable cleavage. Incubation of5′-end-labeled hairpin DNA was for 30 minutes at 55° C., with theindicated components. The products were resolved by denaturingpolyacrylamide gel electrophoresis and the lengths of the products, innucleotides, are indicated. FIG. 8B describes the effect of temperatureon the site of cleavage in the absence of added primer. Reactions wereincubated in the absence of KCl for 10 minutes at the indicatedtemperatures. The lengths of the products, in nucleotides, areindicated.

Surprisingly, cleavage by DNAPTaq requires neither a primer nor dNTPs(See FIG. 8A). Thus, the 5′ nuclease activity can be uncoupled frompolymerization. Nuclease activity requires magnesium ions, thoughmanganese ions can be substituted, albeit with potential changes inspecificity and activity. Neither zinc nor calcium ions support thecleavage reaction. The reaction occurs over a broad temperature range,from 25° C. to 85° C., with the rate of cleavage increasing at highertemperatures.

Still referring to FIG. 8, the primer is not elongated in the absence ofadded dNTPs. However, the primer influences both the site and the rateof cleavage of the hairpin. The change in the site of cleavage (FIG. 8A)apparently results from disruption of a short duplex formed between thearms of the DNA substrate. In the absence of primer, the sequencesindicated by underlining in FIG. 5 could pair, forming an extendedduplex. Cleavage at the end of the extended duplex would release the 11nucleotide fragment seen on the FIG. 8A lanes with no added primer.Addition of excess primer (FIG. 8A, lanes 3 and 4) or incubation at anelevated temperature (FIG. 8B) disrupts the short extension of theduplex and results in a longer 5′ arm and, hence, longer cleavageproducts.

The location of the 3′ end of the primer can influence the precise siteof cleavage. Electrophoretic analysis revealed that in the absence ofprimer (FIG. 8B), cleavage occurs at the end of the substrate duplex(either the extended or shortened form, depending on the temperature)between the first and second base pairs. When the primer extends up tothe base of the duplex, cleavage also occurs one nucleotide into theduplex. However, when a gap of four or six nucleotides exists betweenthe 3′ end of the primer and the substrate duplex, the cleavage site isshifted four to six nucleotides in the 5′ direction.

FIG. 9 describes the kinetics of cleavage in the presence (FIG. 9A) orabsence (FIG. 9B) of a primer oligonucleotide. The reactions were run at55° C. with either 50 mM KCl (FIG. 9A) or 20 mM KCl (FIG. 9B). Thereaction products were resolved by denaturing polyacrylamide gelelectrophoresis and the lengths of the products, in nucleotides, areindicated. “M”, indicating a marker, is a 5′ end-labeled 19-ntoligonucleotide. Under these salt conditions, FIGS. 9A and 9B indicatethat the reaction appears to be about twenty times faster in thepresence of primer than in the absence of primer. This effect on theefficiency may be attributable to proper alignment and stabilization ofthe enzyme on the substrate.

The relative influence of primer on cleavage rates becomes much greaterwhen both reactions are run in 50 mM KCl. In the presence of primer, therate of cleavage increases with KCl concentration, up to about 50 mM.However, inhibition of this reaction in the presence of primer isapparent at 100 mM and is complete at 150 mM KCl. In contrast, in theabsence of primer the rate is enhanced by concentration of KCl up to 20mM, but it is reduced at concentrations above 30 mM. At 50 mM KCl, thereaction is almost completely inhibited. The inhibition of cleavage byKCl in the absence of primer is affected by temperature, being morepronounced at lower temperatures.

Recognition of the 5′ end of the arm to be cut appears to be animportant feature of substrate recognition. Substrates that lack a free5′ end, such as circular M13 DNA, cannot be cleaved under any conditionstested. Even with substrates having defined 5′ arms, the rate ofcleavage by DNAPTaq is influenced by the length of the arm. In thepresence of primer and 50 mM KCl, cleavage of a 5′ extension that is 27nucleotides long is essentially complete within 2 minutes at 55° C. Incontrast, cleavages of molecules with 5′ arms of 84 and 188 nucleotidesare only about 90% and 40% complete after 20 minutes. Incubation athigher temperatures reduces the inhibitory effects of long extensionsindicating that secondary structure in the 5′ arm or a heat-labilestructure in the enzyme may inhibit the reaction. A mixing experiment,run under conditions of substrate excess, shows that the molecules withlong arms do not preferentially tie up the available enzyme innon-productive complexes. These results may indicate that the 5′nuclease domain gains access to the cleavage site at the end of thebifurcated duplex by moving down the 5′ arm from one end to the other.Longer 5′ arms would be expected to have more adventitious secondarystructures (particularly when KCl concentrations are high), which wouldbe likely to impede this movement.

Cleavage does not appear to be inhibited by long 3′ arms of either thesubstrate strand target molecule or pilot nucleic acid, at least up to 2kilobases. At the other extreme, 3′ arms of the pilot nucleic acid asshort as one nucleotide can support cleavage in a primer-independentreaction, albeit inefficiently. Fully paired oligonucleotides do notelicit cleavage of DNA templates during primer extension.

The ability of DNAPTaq to cleave molecules even when the complementarystrand contains only one unpaired 3′ nucleotide may be useful inoptimizing allele-specific PCR. PCR primers that have unpaired 3′ endscould act as pilot oligonucleotides to direct selective cleavage ofunwanted templates during preincubation of potential template-primercomplexes with DNAPTaq in the absence of nucleoside triphosphates.

B. 5′ Nuclease Activities of Other DNAPs

To determine whether other 5′ nucleases in other DNAPs would be suitablefor the present invention, an array of enzymes, several of which werereported in the literature to be free of apparent 5′ nuclease activity,were examined. The ability of these other enzymes to cleave nucleicacids in a structure-specific manner was tested using the hairpinsubstrate shown in FIG. 5 under conditions reported to be optimal forsynthesis by each enzyme.

DNAPEc1 and DNAP Klenow were obtained from Promega; the DNAP ofPyrococcus furious (“Pfu”, Bargseid et al., Strategies 4:34 [1991]) wasfrom Stratagene; the DNAP of Thermococcus litoralis (“Tli”, Vent™(exo-),Perler et al., Proc. Natl. Acad. Sci. USA 89:5577 [1992] was from NewEngland Biolabs; the DNAP of Thermus flavus(“Tfl”, Kaledin et al.,Biokhimiya 46:1576 [1981] was from Epicentre Technologies; and the DNAPof Thermus thermophilus (“Tth”, Carballeira et al., Biotechn., 9:276[1990]; Myers et al., Biochem., 30:7661 (1991)] was from U.S.Biochemicals.

0.5 units of each DNA polymerase was assayed in a 20 μl reaction, usingeither the buffers supplied by the manufacturers for theprimer-dependent reactions, or 10 mM Tris. Cl, pH 8.5, 1.5 mM MgCl₂, and20 mM KCl. Reaction mixtures were at held 72° C. before the addition ofenzyme.

FIG. 10 is an autoradiogram recording the results of these tests. FIG.10A demonstrates reactions of endonucleases of DNAPs of severalthermophilic bacteria. The reactions were incubated at 55° C. for 10minutes in the presence of primer or at 72° C. for 30 minutes in theabsence of primer, and the products were resolved by denaturingpolyacrylamide gel electrophoresis. The lengths of the products, innucleotides, are indicated. FIG. 10B demonstrates endonucleolyticcleavage by the 5′ nuclease of DNAPEc1. The DNAPEc1 and DNAP Klenowreactions were incubated for 5 minutes at 37° C. Note the light band ofcleavage products of 25 and 11 nucleotides in the DNAPEc1 lanes (made inthe presence and absence of primer, respectively). FIG. 8A alsodemonstrates DNAPTaq reactions in the presence (+) or absence (−) ofprimer. These reactions were run in 50 mM and 20 mM KCl, respectively,and were incubated at 55° C. for 10 minutes.

Referring to FIG. 10A, DNAPs from the eubacteria Thermus thermophilusand Thermus flavus cleave the substrate at the same place as DNAPTaq,both in the presence and absence of primer. In contrast, DNAPs from thearchaebacteria Pyrococcus furiosus and Thermococcus litoralis are unableto cleave the substrates endonucleolytically. The DNAPs from Pyrococcusfurious and Thermococcus litoralis share little sequence homology witheubacterial enzymes (Ito et al., Nucl. Acids Res. 19:4045 (1991); Mathuret al., Nucl. Acids. Res. 19:6952 (1991); see also Perler et al.).Referring to FIG. 10B, DNAPEc1 also cleaves the substrate, but theresulting cleavage products are difficult to detect unless the 3′exonuclease is inhibited. The amino acid sequences of the 5′ nucleasedomains of DNAPEc1 and DNAPTaq are about 38% homologous (Gelfand,supra).

The 5′ nuclease domain of DNAPTaq also shares about 19% homology withthe 5′ exonuclease encoded by gene 6 of bacteriophage T7 (Dunn et al.,J. Mol. Biol.,166:477 [1983]). This nuclease, which is not covalentlyattached to a DNAP polymerization domain, is also able to cleave DNAendonucleolytically, at a site similar or identical to the site that iscut by the 5′ nucleases described above, in the absence of addedprimers.

C. Transcleavage

The ability of a 5′ nuclease to be directed to cleave efficiently at anyspecific sequence was demonstrated in the following experiment. Apartially complementary oligonucleotide termed a “pilot oligonucleotide”was hybridized to sequences at the desired point of cleavage. Thenon-complementary part of the pilot oligonucleotide provided a structureanalogous to the 3′ arm of the template (see FIG. 5), whereas the 5′region of the substrate strand became the 5′ arm. A primer was providedby designing the 3′ region of the pilot so that it would fold on itselfcreating a short hairpin with a stabilizing tetra-loop (Antao et al.,Nucl. Acids Res. 19:5901 [1991). Two pilot oligonucleotides are shown inFIG. 11A. Oligonucleotides 19-12 (SEQ ID NO:18), 30-12 (SEQ ID NO:19)and 30-0 (SEQ ID NO:20) are 31, 42 or 30 nucleotides long, respectively.However, oligonucleotides 19-12 (SEQ ID NO:18) and 34-19 (SEQ ID NO:19)have only 19 and 30 nucleotides, respectively, that are complementary todifferent sequences in the substrate strand. The pilot oligonucleotidesare calculated to melt off their complements at about 50° C. (19-12) andabout 75° C. (30-12). Both pilots have 12 nucleotides at their 3′ ends,which act as 3′ arms with base-paired primers attached.

To demonstrate that cleavage could be directed by a pilotoligonucleotide, a single-stranded target DNA with DNAPTaq was incubatedin the presence of two potential pilot oligonucleotides. Thetranscleavage reactions, where the target and pilot nucleic acids arenot covalently linked, includes 0.01 pmoles of single end-labeledsubstrate DNA, 1 unit of DNAPTaq and 5 pmoles of pilot oligonucleotidein a volume of 20 μl of the same buffers. These components were combinedduring a one minute incubation at 95° C., to denature the PCR-generateddouble-stranded substrate DNA, and the temperatures of the reactionswere then reduced to their final incubation temperatures.Oligonucleotides 30-12 and 19-12 can hybridize to regions of thesubstrate DNAs that are 85 and 27 nucleotides from the 5′ end of thetargeted strand.

FIG. 19 shows the complete 206-mer sequence (SEQ ID NO:27). The 206-merwas generated by PCR. The M13/pUC 24-mer reverse sequencing (−48) primerand the M13/pUC sequencing (−47) primer from NEB (catalogue nos. 1233and 1224 respectively) were used (50 pmoles each) with the pGEM3z(f+)plasmid vector (Promega) as template (10 ng) containing the targetsequences. The conditions for PCR were as follows: 50 μM of each dNTPand 2.5 units of Taq DNA polymerase in 100 μl of 20 mM Tris-Cl, pH 8.3,1.5 mM MgCl₂, 50 mM KCl with 0.05% Tween-20 and 0.05% NP-40. Reactionswere cycled 35 times through 95° C. for 45 seconds, 63° C. for 45seconds, then 72° C. for 75 seconds. After cycling, reactions werefinished off with an incubation at 72° C. for 5 minutes. The resultingfragment was purified by electrophoresis through a 6% polyacrylamide gel(29:1 cross link) in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA,visualized by ethidium bromide staining or autoradiography, excised fromthe gel, eluted by passive diffusion, and concentrated by ethanolprecipitation.

Cleavage of the substrate DNA occurred in the presence of the pilotoligonucleotide 19-12 at 50° C. (FIG. 11B, lanes 1 and 7) but not at 75°C. (lanes 4 and 10). In the presence of oligonucleotide 30-12 cleavagewas observed at both temperatures. Cleavage did not occur in the absenceof added oligonucleotides (lanes 3, 6 and 12) or at about 80° C. eventhough at 50° C. adventitious structures in the substrate allowedprimer-independent cleavage in the absence of KCl (FIG. 11B, lane 9). Anon-specific oligonucleotide with no complementarity to the substrateDNA did not direct cleavage at 50° C., either in the absence or presenceof 50 mM KCl (lanes 13 and 14). Thus, the specificity of the cleavagereactions can be controlled by the extent of complementarity to thesubstrate and by the conditions of incubation.

D. Cleavage of RNA

A shortened RNA version of the sequence used in the transcleavageexperiments discussed above was tested for its ability to serve as asubstrate in the reaction. The RNA is cleaved at the expected place, ina reaction that is dependent upon the presence of the pilotoligonucleotide. The RNA substrate, made by T7 RNA polymerase in thepresence of (α-³²P)UTP, corresponds to a truncated version of the DNAsubstrate used in FIG. 11B. Reaction conditions were similar to those inused for the DNA substrates described above, with 50 mM KCl; incubationwas for 40 minutes at 55° C. The pilot oligonucleotide used is termed30-0 (SEQ ID NO:20) and is shown in FIG. 12A.

The results of the cleavage reaction is shown in FIG. 13B. The reactionwas run either in the presence or absence of DNAPTaq or pilotoligonucleotide as indicated in FIG. 12B.

Strikingly, in the case of RNA cleavage, a 3′ arm is not required forthe pilot oligonucleotide. It is very unlikely that this cleavage is dueto previously described RNaseH, which would be expected to cut the RNAin several places along the 30 base-pair long RNA-DNA duplex. The 5′nuclease of DNAPTaq is a structure-specific RNaseH that cleaves the RNAat a single site near the 5′ end of the heteroduplexed region.

It is surprising that an oligonucleotide lacking a 3′ arm is able to actas a pilot in directing efficient cleavage of an RNA target because sucholigonucleotides are unable to direct efficient cleavage of DNA targetsusing native DNAPs. However, some 5′ nucleases of the present invention(for example, clones E, F and G of FIG. 4) can cleave DNA in the absenceof a 3′ arm. In other words, a non-extendable cleavage structure is notrequired for specific cleavage with some 5′ nucleases of the presentinvention derived from thermostable DNA polymerases.

Tests were then conducted to determine whether cleavage of an RNAtemplate by DNAPTaq in the presence of a filly complementary primercould help explain why DNAPTaq is unable to extend a DNA oligonucleotideon an RNA template, in a reaction resembling that of reversetranscriptase. Another thermophilic DNAP, DNAPTth, is able to use RNA asa template, but only in the presence of Mn++, so it was predicted thatthis enzyme would not cleave RNA in the presence of this cation.Accordingly, an RNA molecule was incubated with an appropriate pilotoligonucleotide in the presence of DNAPTaq or DNAPTth, in buffercontaining either Mg++ or Mn++. As expected, both enzymes cleaved theRNA in the presence of Mg++. However, DNAPTaq, but not DNAPTth, degradedthe RNA in the presence of Mn++. It was concluded that the 5′ nucleaseactivities of many DNAPs may contribute to their inability to use RNA astemplates.

Example 2 Generation of 5′ Nucleases from Thermostable DNA Polymerases

Thermostable DNA polymerases were generated which have reduced syntheticactivity, an activity that is an undesirable side-reaction during DNAcleavage in the detection assay of the invention, yet have maintainedthermostable nuclease activity. The result is a thermostable polymerasewhich cleaves nucleic acids DNA with extreme specificity.

Type A DNA polymerases from eubacteria of the genus Thermus shareextensive protein sequence identity (90% in the polymerization domain,using the Lipman-Pearson method in the DNA analysis software fromDNAStar, WI) and behave similarly in both polymerization and nucleaseassays. Therefore, the genes for the DNA polymerase of Thermus aquaticus(DNAPTaq) and Thermus flavus (DNAPTfl) are used as representatives ofthis class. Polymerase genes from other eubacterial organisms, such asThermus thermophilus, Thermus sp., Thermotoga maritima, Thermosiphoafricanus and Bacillus stearothermophilus are equally suitable. The DNApolymerases from these thermophilic organisms are capable of survivingand performing at elevated temperatures, and can thus be used inreactions in which temperature is used as a selection againstnon-specific hybridization of nucleic acid strands.

The restriction sites used for deletion mutagenesis, described below,were chosen for convenience. Different sites situated with similarconvenience are available in the Thermus thermophilus gene and can beused to make similar constructs with other Type A polymerase genes fromrelated organisms.

A. Creation of 5′ Nuclease Constructs

1. Modified DNAPTaq Genes

The first step was to place a modified gene for the Taq DNA polymeraseon a plasmid under control of an inducible promoter. The modified Taqpolymerase gene was isolated as follows: The Taq DNA polymerase gene wasamplified by polymerase chain reaction from genomic DNA from Thermusaquaticus, strain YT-1 (Lawyer et al., supra), using as primers theoligonucleotides described in SEQ ID NOS:13-14. The resulting fragmentof DNA has a recognition sequence for the restriction endonuclease EcoRIat the 5′ end of the coding sequence and a BglII sequence at the 3′ end.Cleavage with BglII leaves a 5′ overhang or “sticky end” that iscompatible with the end generated by BamHI. The PCR-amplified DNA wasdigested with EcoRI and BamHI. The 2512 bp fragment containing thecoding region for the polymerase gene was gel purified and then ligatedinto a plasmid which contains an inducible promoter.

In one embodiment of the invention, the pTTQ18 vector, which containsthe hybrid trp-lac (tac) promoter, was used (Stark, Gene 5:255 [1987])and shown in FIG. 13. The tac promoter is under the control of the E.coli lac repressor. Repression allows the synthesis of the gene productto be suppressed until the desired level of bacterial growth has beenachieved, at which point repression is removed by addition of a specificinducer, isopropyl-β-D-thiogalactopyranoside (IPTG). Such a systemallows the expression of foreign proteins that may slow or preventgrowth of transformants.

Bacterial promoters, such as tac, may not be adequately suppressed whenthey are present on a multiple copy plasmid. If a highly toxic proteinis placed under control of such a promoter, the small amount ofexpression leaking through can be harmful to the bacteria. In anotherembodiment of the invention, another option for repressing synthesis ofa cloned gene product was used. The non-bacterial promoter, frombacteriophage T7, found in the plasmid vector series pET-3 was used toexpress the cloned mutant Taq polymerase genes (FIG. 15; Studier andMoffatt, J. Mol. Biol., 189:113 [1986]). This promoter initiatestranscription only by T7 RNA polymerase. In a suitable strain, such asBL21(DE3)pLYS, the gene for this RNA polymerase is carried on thebacterial genome under control of the lac operator. This arrangement hasthe advantage that expression of the multiple copy gene (on the plasmid)is completely dependent on the expression of T7 RNA polymerase, which iseasily suppressed because it is present in a single copy.

For ligation into the pTTQ18 vector (FIG. 13), the PCR product DNAcontaining the Taq polymerase coding region (mutTaq, clone 4B, SEQ IDNO:21) was digested with EcoRI and BglII and this fragment was ligatedunder standard “sticky end” conditions (Sambrook et al. MolecularCloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp.1.63-1.69 [1989]) into the EcoRI and BamHI sites of the plasmid vectorpTTQ18. Expression of this construct yields a translational fusionproduct in which the first two residues of the native protein (Met-Arg)are replaced by three from the vector (Met-Asn-Ser), but the remainderof the natural protein would not change. The construct was transformedinto the JM109 strain of E. coli and the transformants were plated underincompletely repressing conditions that do not permit growth of bacteriaexpressing the native protein. These plating conditions allow theisolation of genes containing pre-existing mutations, such as those thatresult from the infidelity of Taq polymerase during the amplificationprocess.

Using this amplification/selection protocol, a clone (depicted in FIG.3B) containing a mutated Taq polymerase gene (mutTaq, clone 3B) wasisolated. The mutant was first detected by its phenotype, in whichtemperature-stable 5′ nuclease activity in a crude cell extract wasnormal, but polymerization activity was almost absent (approximatelyless than 1% of wild type Taq polyrnerase activity).

DNA sequence analysis of the recombinant gene showed that it had changesin the polymerase domain resulting in two amino acid substitutions: an Ato G change at nucleotide position 1394 causes a Glu to Gly change atamino acid position 465 (numbered according to the natural nucleic andamino acid sequences, SEQ ID NOS:1 and 4) and another A to G change atnucleotide position 2260 causes a Gln to Arg change at amino acidposition 754. Because the Gln to Gly mutation is at a nonconservedposition and because the Glu to Arg mutation alters an amino acid thatis conserved in virtually all of the known Type A polymerases, thislatter mutation is most likely the one responsible for curtailing thesynthesis activity of this protein. The nucleotide sequence for the FIG.3B construct is given in SEQ ID NO:21. The enzyme encoded by thissequence is referred to as Cleavase® A/G.

Subsequent derivatives of DNAPTaq constructs were made from the mutTaqgene, thus, they all bear these amino acid substitutions in addition totheir other alterations, unless these particular regions were deleted.These mutated sites are indicated by black boxes at these locations inthe diagrams in FIG. 3. In FIG. 3, the designation “3′ Exo” is used toindicate the location of the 3′ exonuclease activity associated withType A polymerases which is not present in DNAPTaq. All constructsexcept the genes shown in FIGS. 3E, F and G were made in the pTTQ18vector.

The cloning vector used for the genes in FIGS. 3E and F was from thecommercially available pET-3 series, described above. Though this vectorseries has only a BamHI site for cloning downstream of the T7 promoter,the series contains variants that allow cloning into any of the threereading frames. For cloning of the PCR product described above, thevariant called pET-3c was used (FIG. 14). The vector was digested withBamHI, dephosphorylated with calf intestinal phosphatase, and the stickyends were filled in using the Klenow fragment of DNAPEc1 and dNTPs. Thegene for the mutant Taq DNAP shown in FIG. 3B (mutTaq, clone 3B) wasreleased from pTTQ18 by digestion with EcoRI and SalI, and the “stickyends” were filled in as was done with the vector. The fragment wasligated to the vector under standard blunt-end conditions (Sambrook etal., Molecular Cloning, supra), the construct was transformed into theBL21(DE3)pLYS strain of E. coli, and isolates were screened to identifythose that were ligated with the gene in the proper orientation relativeto the promoter. This construction yields another translational fusionproduct, in which the first two amino acids of DNAPTaq (Met-Arg) arereplaced by 13 from the vector plus two from the PCR primer(Met-Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg-Ile-Asn-Ser) (SEQ IDNO:24).

In these experiments, the goal was to generate enzymes that lacked theability to synthesize DNA, but retained the ability to cleave nucleicacids with a 5′ nuclease activity. The act of primed, templatedsynthesis of DNA is actually a coordinated series of events, so it ispossible to disable DNA synthesis by disrupting one event while notaffecting the others. These steps include, but are not limited to,primer recognition and binding, dNTP binding and catalysis of theinter-nucleotide phosphodiester bond. Some of the amino acids in thepolymerization domain of DNAPEcI have been linked to these functions,but the precise mechanisms are as yet poorly defined.

One way of destroying the polymerizing ability of a DNA polymerase is todelete all or part of the gene segment that encodes that domain for theprotein, or to otherwise render the gene incapable of making a completepolymerization domain. Individual mutant enzymes may differ from eachother in stability and solubility both inside and outside cells. Forinstance, in contrast to the 5′ nuclease domain of DNAPEcI, which can bereleased in an active form from the polymerization domain by gentleproteolysis (Setlow and Kornberg, J. Biol. Chem., 247:232 [1972]), theThermus nuclease domain, when treated similarly, becomes less solubleand the cleavage activity is often lost.

Using the mutant gene shown in FIG. 3B as starting material, severaldeletion constructs were created. All cloning technologies were standard(Sambrook et al., supra) and are summarized briefly, as follows:

FIG. 3C: The mutTaq construct was digested with PstI, which cuts oncewithin the polymerase coding region, as indicated, and cuts immediatelydownstream of the gene in the multiple cloning site of the vector. Afterrelease of the fragment between these two sites, the vector wasre-ligated, creating an 894-nucleotide deletion, and bringing into framea stop codon 40 nucleotides downstream of the junction. The nucleotidesequence of this 5′ nuclease (clone 4C) is given in SEQ ID NO:9.

FIG. 3D: The mutTaq construct was digested with NheI, which cuts once inthe gene at position 2047. The resulting four-nucleotide 5′ overhangingends were filled in, as described above, and the blunt ends werere-ligated. The resulting four-nucleotide insertion changes the readingframe and causes termination of translation ten amino acids downstreamof the mutation. The nucleotide sequence of this 5′ nuclease (clone 3D)is given in SEQ ID NO:10.

FIG. 3E: The entire mutTaq gene was cut from pTTQ18 using EcoRI and SalIand cloned into pET-3c, as described above. This clone was digested withBstXI and XcmI, at unique sites that are situated as shown in FIG. 3E.The DNA was treated with the Klenow fragment of DNAPEc1 and dNTPs, whichresulted in the 3′ overhangs of both sites being trimmed to blunt ends.These blunt ends were ligated together, resulting in an out-of-framedeletion of 1540 nucleotides. An in-frame termination codon occurs 18triplets past the junction site. The nucleotide sequence of this 5′nuclease (clone 3E) is given in SEQ ID NO:11, with the appropriateleader sequence given in SEQ ID NO:25. It is also referred to asCleavase® BX.

FIG. 3F: The entire mutTaq gene was cut from pTTQ18 using EcoRI and SalIand cloned into pET-3c, as described above. This clone was digested withBstXI and BamHI, at unique sites that are situated as shown in thediagram. The DNA was treated with the Klenow fragment of DNAPEc1 anddNTPs, which resulted in the 3′ overhang of the BstXI site being trimmedto a blunt end, while the 5′ overhang of the BamHI site was filled in tomake a blunt end. These ends were ligated together, resulting in anin-frame deletion of 903 nucleotides. The nucleotide sequence of the 5′nuclease (clone 3F) is given in SEQ ID NO:12. It is also referred to asCleavase® BB.

FIG. 3G: This polymerase is a variant of that shown in FIG. 4E. It wascloned in the plasmid vector pET-21 (Novagen). The non-bacterialpromoter from bacteriophage T7, found in this vector, initiatestranscription only by T7 RNA polymerase. See Studier and Moffatt, supra.In a suitable strain, such as (DES)pLYS, the gene for this RNApolymerase is carried on the bacterial genome under control of the lacoperator. This arrangement has the advantage that expression of themultiple copy gene (on the plasmid) is completely dependent on theexpression of T7 RNA polymerase, which is easily suppressed because itis present in a single copy. Because the expression of these mutantgenes is under this tightly controlled promoter, potential problems oftoxicity of the expressed proteins to the host cells are less of aconcern.

The pET-21 vector also features a “His*Tag”, a stretch of sixconsecutive histidine residues that are added on the carboxy terminus ofthe expressed proteins. The resulting proteins can then be purified in asingle step by metal chelation chromatography, using a commerciallyavailable (Novagen) column resin with immobilized Ni⁺⁺ ions. The 2.5 mlcolumns are reusable, and can bind up to 20 mg of the target proteinunder native or denaturing (guanidine*HCl or urea) conditions.

E. coli (DES)pLYS cells are transformed with the constructs describedabove using standard transformation techniques, and used to inoculate astandard growth medium (e.g., Luria-Bertani broth). Production of T7 RNApolymerase is induced during log phase growth by addition of IPTG andincubated for a further 12 to 17 hours. Aliquots of culture are removedboth before and after induction and the proteins are examined bySDS-PAGE. Staining with Coomassie Blue allows visualization of theforeign proteins if they account for about 3-5% of the cellular proteinand do not co-migrate with any of the major protein bands. Proteins thatco-migrate with major host protein must be expressed as more than 10% ofthe total protein to be seen at this stage of analysis.

Some mutant proteins are sequestered by the cells into inclusion bodies.These are granules that form in the cytoplasm when bacteria are made toexpress high levels of a foreign protein, and they can be purified froma crude lysate, and analyzed by SDS-PAGE to determine their proteincontent. If the cloned protein is found in the inclusion bodies, it mustbe released to assay the cleavage and polymerase activities. Differentmethods of solubilization may be appropriate for different proteins, anda variety of methods are known (See e.g., Builder & Ogez, U.S. Pat. No.4,511,502 (1985); Olson, U.S. Pat. No. 4,518,526 (1985); Olson & Pai,U.S. Pat. No. 4,511,503 (1985); and Jones et al., U.S. Pat. No.4,512,922 (1985), all of which are hereby incorporated by reference).

The solubilized protein is then purified on the Ni⁺⁺ column as describedabove, following the manufacturers instructions (Novagen). The washedproteins are eluted from the column by a combination of imidazolecompetitor (1 M) and high salt (0.5 M NaCl), and dialyzed to exchangethe buffer and to allow denature proteins to refold. Typical recoveriesresult in approximately 20 μg of specific protein per ml of startingculture. The DNAP mutant is referred to as the CLEAVASE BN nuclease andthe sequence is given in SEQ ID NO:26 (the amino acid sequence of theCLEAVASE BN nuclease is obtained by translating the DNA sequence of SEQID NO:26).

2. Modified DNAPTfl Gene

The DNA polymerase gene of Thermus flavus was isolated from the “T.flavus” AT-62 strain obtained from the American Type Tissue Collection(ATCC 33923). This strain has a different restriction map then does theT. flavus strain used to generate the sequence published by Akhmetzjanovand Vakhitov, supra. The published sequence is listed as SEQ ID NO:2. Nosequence data has been published for the DNA polymerase gene from theAT-62 strain of T. flavus.

Genomic DNA from T. flavus was amplified using the same primers used toamplify the T. aquaticus DNA polymerase gene (SEQ ID NOS:13-14). Theapproximately 2500 base pair PCR fragment was digested with EcoRI andBamHI. The over-hanging ends were made blunt with the Klenow fragment ofDNAPEc1 and dNTPs. The resulting approximately 1800 base pair fragmentcontaining the coding region for the N-terminus was ligated into pET-3c,as described above. This construct, clone 4B, is depicted in FIG. 4B.The wild type T. flavus DNA polymerase gene is depicted in FIG. 4A. The4B clone has the same leader amino acids as do the DNAPTaq clones 4E andF which were cloned into pET-3c; it is not known precisely wheretranslation termination occurs, but the vector has a strongtranscription termination signal immediately downstream of the cloningsite.

B. Growth and Induction of Transformed Cells

Bacterial cells were transformed with the constructs described aboveusing standard transformation techniques and used to inoculate 2 mls ofa standard growth medium (e.g., Luria-Bertani broth). The resultingcultures were incubated as appropriate for the particular strain used,and induced if required for a particular expression system. For all ofthe constructs depicted in FIGS. 3 and 4, the cultures were grown to anoptical density (at 600 nm wavelength) of 0.5 OD.

To induce expression of the cloned genes, the cultures were brought to afinal concentration of 0.4 mM IPTG and the incubations were continuedfor 12 to 17 hours. Then, 50 μl aliquots of each culture were removedboth before and after induction and were combined with 20 μl of astandard gel loading buffer for sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE). Subsequent staining with Coomassie Blue(Sambrook et al., supra) allows visualization of the foreign proteins ifthey account for about 3-5% of the cellular protein and do notco-migrate with any of the major E. coli protein bands. Proteins that doco-migrate with a major host protein must be expressed as more than 10%of the total protein to be seen at this stage of analysis.

C. Heat Lysis and Fractionation

Expressed thermostable proteins (i.e., the 5′ nucleases), were isolatedby heating crude bacterial cell extracts to cause denaturation andprecipitation of the less stable E. coli proteins. The precipitated E.coli proteins were then, along with other cell debris, removed bycentrifugation. Then, 1.7 mls of the culture were pelleted bymicrocentrifugation at 12,000 to 14,000 rpm for 30 to 60 seconds. Afterremoval of the supernatant, the cells were resuspended in 400 μl ofbuffer A (50 mM Tris-HCl, pH 7.9, 50 mM dextrose, 1 mM EDTA),re-centrifuged, then resuspended in 80 μl of buffer A with 4 mg/mllysozyme. The cells were incubated at room temperature for 15 minutes,then combined with 80 μl of buffer B (10 mM Tris-HCl, pH 7.9, 50 mM KCl,1 mM EDTA, 1 mM PMSF, 0.5% Tween-20, 0.5% Nonidet-P40).

This mixture was incubated at 75° C. for 1 hour to denature andprecipitate the host proteins. This cell extract was centrifuged at14,000 rpm for 15 minutes at 4° C., and the supernatant was transferredto a fresh tube. An aliquot of 0.5 to 1 μl of this supernatant was useddirectly in each test reaction, and the protein content of the extractwas determined by subjecting 7 μl to electrophoretic analysis, as above.The native recombinant Taq DNA polymerase (Engelke, Anal. Biochem.,191:396 [1990]), and the double point mutation protein shown in FIG. 3Bare both soluble and active at this point.

The foreign protein may not be detected after the heat treatments due tosequestration of the foreign protein by the cells into inclusion bodies.These are granules that form in the cytoplasm when bacteria are made toexpress high levels of a foreign protein, and they can be purified froma crude lysate, and analyzed SDS PAGE to determine their proteincontent. Many methods have been described in the literature, and oneapproach is described below.

D. Isolation and Solubilization of Inclusion Bodies

A small culture was grown and induced as described above. A 1.7 mlaliquot was pelleted by brief centrifugation, and the bacterial cellswere resuspended in 100 μl of Lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mMEDTA, 100 mM NaCl). Then, 2.5 μl of 20 mM PMSF were added for a finalconcentration of 0.5 mM, and lysozyme was added to a concentration of1.0 mg/ml. The cells were incubated at room temperature for 20 minutes,deoxycholic acid was added to 1 mg/ml (1 μl of 100 mg/ml solution), andthe mixture was further incubated at 37° C. for about 15 minutes oruntil viscous. DNAse I was added to 10 μg/ml and the mixture wasincubated at room temperature for about 30 minutes or until it was nolonger viscous.

From this mixture the inclusion bodies were collected by centrifugationat 14,000 rpm for 15 minutes at 4° C., and the supernatant wasdiscarded. The pellet was resuspended in 100 μl of lysis buffer with 10mM EDTA (pH 8.0) and 0.5% Triton X-100. After 5 minutes at roomtemperature, the inclusion bodies were pelleted as before, and thesupernatant was saved for later analysis. The inclusion bodies wereresuspended in 50 μl of distilled water, and 5 μl was combined with SDSgel loading buffer (which dissolves the inclusion bodies) and analyzedelectrophoretically, along with an aliquot of the supernatant.

If the cloned protein is found in the inclusion bodies, it may bereleased to assay the cleavage and polymerase activities and the methodof solubilization must be compatible with the particular activity.Different methods of solubilization may be appropriate for differentproteins, and a variety of methods are discussed in Molecular Cloning(Sambrook et al., supra). The following is an adaptation used forseveral of the isolates used in the development of the presentinvention.

Twenty μl of the inclusion body-water suspension were pelleted bycentrifugation at 14,000 rpm for 4 minutes at room temperature, and thesupernatant was discarded. To further wash the inclusion bodies, thepellet was resuspended in 20 μl of lysis buffer with 2M urea, andincubated at room temperature for one hour. The washed inclusion bodieswere then resuspended in 2 μl of lysis buffer with 8 M urea; thesolution clarified visibly as the inclusion bodies dissolved.Undissolved debris was removed by centrifugation at 14,000 rpm for 4minutes at room temperature, and the extract supernatant was transferredto a fresh tube.

To reduce the urea concentration, the extract was diluted into KH₂PO₄. Afresh tube was prepared containing 180 μl of 50 mM KH₂PO₄, pH 9.5, 1 mMEDTA and 50 mM NaCl. A 2 μl aliquot of the extract was added andvortexed briefly to mix. This step was repeated until all of the extracthad been added for a total of 10 additions. The mixture was allowed tosit at room temperature for 15 minutes, during which time someprecipitate often forms. Precipitates were removed by centrifugation at14,000 rpm, for 15 minutes at room temperature, and the supernatant wastransferred to a fresh tube. To the 200 μl of protein in the KH₂PO₄solution, 140-200 μl of saturated (NH₄)₂SO₄ were added, so that theresulting mixture was about 41% to 50% saturated (NH₄)₂SO₄. The mixturewas chilled on ice for 30 minutes to allow the protein to precipitate,and the protein was then collected by centrifugation at 14,000 rpm, for4 minutes at room temperature. The supernatant was discarded, and thepellet was dissolved in 20 μl Buffer C(20 mM HEPES, pH 7.9, 1 mM EDTA,0.5% PMSF, 25 mM KCl and 0.5% each of Tween-20 and Nonidet P 40). Theprotein solution was centrifuged again for 4 minutes to pellet insolublematerials, and the supernatant was removed to a fresh tube. The proteincontents of extracts prepared in this manner were visualized byresolving 1-4 μl by SDS-PAGE; 0.5 to 1 μl of extract was tested in thecleavage and polymerization assays as described.

E. Protein Analysis for Presence of Nuclease and Synthetic Activity

The 5′ nucleases described above and shown in FIGS. 3 and 4 wereanalyzed by the following methods.

1. Structure Specific Nuclease Assay

A candidate modified polymerase is tested for 5′ nuclease activity byexamining its ability to catalyze structure-specific cleavages. By theterm “cleavage structure” as used herein, is meant a nucleic acidstructure which is a substrate for cleavage by the 5′ nuclease activityof a DNAP.

The polymerase is exposed to test complexes that have the structuresshown in FIG. 15. Testing for 5′ nuclease activity involves threereactions: 1) a primer-directed cleavage (FIG. 15B) is performed becauseit is relatively insensitive to variations in the salt concentration ofthe reaction and can, therefore, be performed in whatever soluteconditions the modified enzyme requires for activity; this is generallythe same conditions preferred by unmodified polymerases; 2) a similarprimer-directed cleavage is performed in a buffer which permitsprimer-independent cleavage (i.e., a low salt buffer), to demonstratethat the enzyme is viable under these conditions; and 3) aprimer-independent cleavage (FIG. 15A) is performed in the same low saltbuffer.

The bifurcated duplex is formed between a substrate strand and atemplate strand as shown in FIG. 15. By the term “substrate strand” asused herein, is meant that strand of nucleic acid in which the cleavagemediated by the 5′ nuclease activity occurs. The substrate strand isalways depicted as the top strand in the bifurcated complex which servesas a substrate for 5′ nuclease cleavage (FIG. 15). By the term “templatestrand” as used herein, is meant the strand of nucleic acid which is atleast partially complementary to the substrate strand and which annealsto the substrate strand to form the cleavage structure. The templatestrand is always depicted as the bottom strand of the bifurcatedcleavage structure (FIG. 15). If a primer (a short oligonucleotide of 19to 30 nucleotides in length) is added to the complex, as whenprimer-dependent cleavage is to be tested, it is designed to anneal tothe 3′ arm of the template strand (FIG. 15B). Such a primer would beextended along the template strand if the polymerase used in thereaction has synthetic activity.

The cleavage structure may be made as a single hairpin molecule, withthe 3′ end of the target and the 5′ end of the pilot joined as a loop asshown in FIG. 15E. A primer oligonucleotide complementary to the 3′ armis also required for these tests so that the enzyme's sensitivity to thepresence of a primer may be tested.

Nucleic acids to be used to form test cleavage structures can bechemically synthesized, or can be generated by standard recombinant DNAtechniques. By the latter method, the hairpin portion of the moleculecan be created by inserting into a cloning vector duplicate copies of ashort DNA segment, adjacent to each other but in opposing orientation.The double-stranded fragment encompassing this inverted repeat, andincluding enough flanking sequence to give short (about 20 nucleotides)unpaired 5′ and 3′ arms, can then be released from the vector byrestriction enzyme digestion, or by PCR performed with an enzyme lackinga 5′ exonuclease (e.g., the Stoffel fragment of AMPLITAQ DNA polymerase,Vent™ DNA polymerase).

The test DNA can be labeled on either end, or internally, with either aradioisotope, or with a non-isotopic tag. Whether the hairpin DNA is asynthetic single strand or a cloned double strand, the DNA is heatedprior to use to melt all duplexes. When cooled on ice, the structuredepicted in FIG. 16E is formed, and is stable for sufficient time toperform these assays.

To test for primer-directed cleavage (Reaction 1), a detectable quantityof the test molecule (typically 1-100 fmol of ³²P-labeled hairpinmolecule) and a 10 to 100-fold molar excess of primer are placed in abuffer known to be compatible with the test enzyme. For Reaction 2,where primer-directed cleavage is performed under condition which allowprimer-independent cleavage, the same quantities of molecules are placedin a solution that is the same as the buffer used in Reaction 1regarding pH, enzyme stabilizers (e.g., bovine serum albumin, nonionicdetergents, gelatin) and reducing agents (e.g., dithiothreitol,2-mercaptoethanol) but that replaces any monovalent cation salt with 20mM KCl; 20 mM KCl is the demonstrated optimum for primer-independentcleavage. Buffers for enzymes, such as DNAPEc 1, that usually operate inthe absence of salt are not supplemented to achieve this concentration.To test for primer-independent cleavage (Reaction 3) the same quantityof the test molecule, but no primer, are combined under the same bufferconditions used for Reaction 2.

All three test reactions are then exposed to enough of the enzyme thatthe molar ratio of enzyme to test complex is approximately 1:1. Thereactions are incubated at a range of temperatures up to, but notexceeding, the temperature allowed by either the enzyme stability or thecomplex stability, whichever is lower, up to 80° C. for enzymes fromthermophiles, for a time sufficient to allow cleavage (10 to 60minutes). The products of Reactions 1, 2 and 3 are resolved bydenaturing polyacrylamide gel electrophoresis, and visualized byautoradiography or by a comparable method appropriate to the labelingsystem used. Additional labeling systems include chemiluminescencedetection, silver or other stains, blotting and probing and the like.The presence of cleavage products is indicated by the presence ofmolecules which migrate at a lower molecular weight than does theuncleaved test structure. These cleavage products indicate that thecandidate polymerase has structure-specific 5′ nuclease activity.

To determine whether a modified DNA polymerase has substantially thesame 5′ nuclease activity as that of the native DNA polymerase, theresults of the above-described tests are compared with the resultsobtained from these tests performed with the native DNA polymerase. By“substantially the same 5′ nuclease activity” it is meant that themodified polymerase and the native polymerase will both cleave testmolecules in the same manner. It is not necessary that the modifiedpolymerase cleave at the same rate as the native DNA polymerase.

Some enzymes or enzyme preparations may have other associated orcontaminating activities that may be functional under the cleavageconditions described above and that may interfere with 5′ nucleasedetection. Reaction conditions can be modified in consideration of theseother activities, to avoid destruction of the substrate, or othermasking of the 5′ nuclease cleavage and its products. For example, theDNA polymerase I of E. coli (Pol I), in addition to its polymerase and5′ nuclease activities, has a 3′ exonuclease that can degrade DNA in a3′ to 5′ direction. Consequently, when the molecule in FIG. 15E isexposed to this polymerase under the conditions described above, the 3′exonuclease quickly removes the unpaired 3′ arm, destroying thebifurcated structure required of a substrate for the 5′ exonucleasecleavage and no cleavage is detected. The true ability of Pol I tocleave the structure can be revealed if the 3′ exonuclease is inhibitedby a change of conditions (e.g., pH), mutation, or by addition of acompetitor for the activity. Addition of 500 pmoles of a single-strandedcompetitor oligonucleotide, unrelated to the FIG. 15E structure, to thecleavage reaction with Pol I effectively inhibits the digestion of the3′ arm of the FIG. 15E structure without interfering with the 5′exonuclease release of the 5′ arm. The concentration of the competitoris not critical, but should be high enough to occupy the 3′ exonucleasefor the duration of the reaction.

Similar destruction of the test molecule may be caused by contaminantsin the candidate polymerase preparation. Several sets of the structurespecific nuclease reactions may be performed to determine the purity ofthe candidate nuclease and to find the window between under and overexposure of the test molecule to the polymerase preparation beinginvestigated.

The above described modified polymerases were tested for 5′ nucleaseactivity as follows: Reaction 1 was performed in a buffer of 10 mMTris-Cl, pH 8.5 at 20° C., 1.5 mM MgCl₂ and 50 mM KCl and in Reaction 2the KCl concentration was reduced to 20 mM. In Reactions 1 and 2, 10fmoles of the test substrate molecule shown in FIG. 15E were combinedwith 1 pmole of the indicated primer and 0.5 to 1.0 μl of extractcontaining the modified polymerase (prepared as described above). Thismixture was then incubated for 10 minutes at 55° C. For all of themutant polymerases tested these conditions were sufficient to givecomplete cleavage. When the molecule shown in FIG. 15E was labeled atthe 5′ end, the released 5′ fragment, 25 nucleotides long, wasconveniently resolved on a 20% polyacrylamide gel (19:1 cross-linked)with 7 M urea in a buffer containing 45 mM Tris-borate pH 8.3, 1.4 mMEDTA. Clones 3C-F and 4B exhibited structure-specific cleavagecomparable to that of the unmodified DNA polymerase. Additionally,clones 3E, 3F and 3G have the added ability to cleave DNA in the absenceof a 3′ arm as discussed above. Representative cleavage reactions areshown in FIG. 16.

For the reactions shown in FIG. 16, the mutant polymerase clones 3E (Taqmutant) and 4B (Tfl mutant) were examined for their ability to cleavethe hairpin substrate molecule shown in FIG. 15E. The substrate moleculewas labeled at the 5′ terminus with ³²P. Ten fmoles of heat-denatured,end-labeled substrate DNA and 0.5 units of DNAPTaq (lane 1) or 0.5 μl of3E or 4B extract (FIG. 16, lanes 2-7, extract was prepared as describedabove) were mixed together in a buffer containing 10 mM Tris-Cl, pH 8.5,50 mM KCl and 1.5 mM MgCl₂. The final reaction volume was 10 μl.Reactions shown in lanes 4 and 7 contain in addition 50 μM of each dNTP.Reactions shown in lanes 3, 4, 6 and 7 contain 0.2 μM of the primeroligonucleotide (complementary to the 3′ arm of the substrate and shownin FIG. 15E). Reactions were incubated at 55° C. for 4 minutes.Reactions were stopped by the addition of 8 μl of 95% formamidecontaining 20 mM EDTA and 0.05% marker dyes per 10 μl reaction volume.Samples were then applied to 12% denaturing acrylamide gels. Followingelectrophoresis, the gels were autoradiographed. FIG. 16 shows thatclones 3E and 4B exhibit cleavage activity similar to that of the nativeDNAPTaq. Note that some cleavage occurs in these reactions in theabsence of the primer. When long hairpin structure, such as the one usedhere (FIG. 15E), are used in cleavage reactions performed in bufferscontaining 50 mM KCl a low level of primer-independent cleavage is seen.Higher concentrations of KCl suppress, but do not eliminate, thisprimer-independent cleavage under these conditions.

2. Assay for Synthetic Activity

The ability of the modified enzyme or proteolytic fragments is assayedby adding the modified enzyme to an assay system in which a primer isannealed to a template and DNA synthesis is catalyzed by the addedenzyme. Many standard laboratory techniques employ such an assay. Forexample, nick translation and enzymatic sequencing involve extension ofa primer along a DNA template by a polymerase molecule.

In a preferred assay for determining the synthetic activity of amodified enzyme an oligonucleotide primer is annealed to asingle-stranded DNA template (e.g., bacteriophage M13 DNA), and theprimer/template duplex is incubated in the presence of the modifiedpolymerase in question, deoxynucleoside triphosphates (dNTPs) and thebuffer and salts known to be appropriate for the unmodified or nativeenzyme. Detection of either primer extension (by denaturing gelelectrophoresis) or dNTP incorporation (by acid precipitation orchromatography) is indicative of an active polymerase. A label, eitherisotopic or non-isotopic, is preferably included on either the primer oras a dNTP to facilitate detection of polymerization products. Syntheticactivity is quantified as the amount of free nucleotide incorporatedinto the growing DNA chain and is expressed as amount incorporated perunit of time under specific reaction conditions.

Representative results of an assay for synthetic activity is shown inFIG. 17. The synthetic activity of the mutant DNAPTaq clones 3B-F wastested as follows: A master mixture of the following buffer was made:1.2× PCR buffer (1× PCR buffer contains 50 mM KCl, 1.5 mM MgCl₂, 10 mMTris-Cl, pH 8.5 and 0.05% each Tween 20 and Nonidet P40), 50 μM each ofdGTP, dATP and dTTP, 5 μM dCTP and 0.125 μM α-³²P-dCTP at 600 Ci/mmol.Before adjusting this mixture to its final volume, it was divided intotwo equal aliquots. One received distilled water up to a volume of 50 μlto give the concentrations above. The other received 5 μg ofsingle-stranded M13mp18 DNA (approximately 2.5 pmol or 0.05 μM finalconcentration) and 250 pmol of M13 sequencing primer (5 μM finalconcentration) and distilled water to a final volume of 50 μl. Eachcocktail was warmed to 75° C. for 5 minutes and then cooled to roomtemperature. This allowed the primers to anneal to the DNA in theDNA-containing mixtures.

For each assay, 4 μl of the cocktail with the DNA was combined with 1 μlof the mutant polymerase, prepared as described, or 1 unit of DNAPTaq(Perkin Elmer) in 1 μl of dH₂O. A “no DNA” control was done in thepresence of the DNAPTaq (FIG. 17, lane 1), and a “no enzyme” control wasdone using water in place of the enzyme (lane 2). Each reaction wasmixed, then incubated at room temperature (approx. 22° C.) for 5minutes, then at 55° C. for 2 minutes, then at 72° C. for 2 minutes.This step incubation was done to detect polymerization in any mutantsthat might have optimal temperatures lower than 72° C. After the finalincubation, the tubes were spun briefly to collect any condensation andwere placed on ice. One μl of each reaction was spotted at an origin 1.5cm from the bottom edge of a polyethyleneimine (PEI) cellulose thinlayer chromatography plate and allowed to dry. The chromatography platewas run in 0.75 M NaH₂PO₄, pH 3.5, until the buffer front had runapproximately 9 cm from the origin. The plate was dried, wrapped inplastic wrap, marked with luminescent ink, and exposed to X-ray film.Incorporation was detected as counts that stuck where originallyspotted, while the unincorporated nucleotides were carried by the saltsolution from the origin.

Comparison of the locations of the counts with the two control lanesconfirmed the lack of polymerization activity in the mutantpreparations. Among the modified DNAPTaq clones, only clone 3B retainsany residual synthetic activity as shown in FIG. 17.

Example 3 5′ Nucleases Derived from Thermostable DNA Polymerases canCleave Short Hairpin Structures with Specificity

The ability of the 5′ nucleases to cleave hairpin structures to generatea cleaved hairpin structure suitable as a detection molecule wasexamined. The structure and sequence of the hairpin test molecule isshown in FIG. 18A (SEQ ID NO:15). The oligonucleotide (labeled “primer”in FIG. 18A, SEQ ID NO:22) is shown annealed to its complementarysequence on the 3′ arm of the hairpin test molecule. The hairpin testmolecule was single-end labeled with ³²P using a labeled T7 promoterprimer in a polymerase chain reaction. The label is present on the 5′arm of the hairpin test molecule and is represented by the star in FIG.18A.

The cleavage reaction was performed by adding 10 fmoles ofheat-denatured, end-labeled hairpin test molecule, 0.2 μM of the primeroligonucleotide (complementary to the 3′ arm of the hairpin), 50 μM ofeach dNTP and 0.5 units of DNAPTaq (Perkin Elmer) or 0.5 μl of extractcontaining a 5′ nuclease (prepared as described above) in a total volumeof 10 μl in a buffer containing 10 mM Tris-Cl, pH 8.5, 50 mM KCl and 1.5mM MgCl₂. Reactions shown in lanes 3, 5 and 7 were run in the absence ofdNTPs.

Reactions were incubated at 55° C. for 4 minutes. Reactions were stoppedat 55° C. by the addition of 8 μl of 95% formamide with 20 mM EDTA and0.05% marker dyes per 10 μl reaction volume. Samples were not heatedbefore loading onto denaturing polyacrylamide gels (10% polyacrylamide,19:1 crosslinking, 7 M urea, 89 mM Tris-borate, pH 8.3, 2.8 mM EDTA).The samples were not heated to allow for the resolution ofsingle-stranded and re-duplexed uncleaved hairpin molecules.

FIG. 18B shows that altered polymerases lacking any detectable syntheticactivity cleave a hairpin structure when an oligonucleotide is annealedto the single-stranded 3′ arm of the hairpin to yield a single speciesof cleaved product (FIG. 18B, lanes 3 and 4). 5′ nucleases, such asclone 3D, shown in lanes 3 and 4, produce a single cleaved product evenin the presence of dNTPs. 5′ nucleases that retain a residual amount ofsynthetic activity (less than 1% of wild type activity) produce multiplecleavage products as the polymerase can extend the oligonucleotideannealed to the 3′ arm of the hairpin thereby moving the site ofcleavage (clone 3B, lanes 5 and 6). Native DNATaq produces even morespecies of cleavage products than do mutant polymerases retainingresidual synthetic activity and additionally converts the hairpinstructure to a double-stranded form in the presence of dNTPs due to thehigh level of synthetic activity in the native polymerase (FIG. 18B,lane 8).

Example 4 Cleavage of Linear Nucleic Acid Substrates

From the above, it should be clear that native (i.e., “wild type”)thermostable DNA polymerases are capable of cleaving hairpin structuresin a specific manner and that this discovery can be applied with successto a detection assay. In this example, the mutant DNAPs of the presentinvention are tested against three different cleavage structures shownin FIG. 20A. Structure 1 in FIG. 20A is simply single stranded 206-mer(the preparation and sequence information for which was discussed inExample 1C). Structures 2 and 3 are duplexes; structure 2 is the samehairpin structure as shown in FIG. 11A (bottom), while structure 3 hasthe hairpin portion of structure 2 removed.

The cleavage reactions comprised 0.01 pmoles of the resulting substrateDNA, and 1 pmole of pilot oligonucleotide in a total volume of 10 μl of10 mM Tris-Cl, pH 8.3, 100 mM KCl, 1 mM MgCl₂. Reactions were incubatedfor 30 minutes at 55° C., and stopped by the addition of 8 μl of 95%formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to75° C. for 2 minutes immediately before electrophoresis through a 10%polyacrylamide gel (19:1 cross link), with 7M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA.

The results were visualized by autoradiography and are shown in FIG. 20Bwith the enzymes indicated as follows: I is native Taq DNAP; II isnative Tfl DNAP; III is CLEAVASE BX shown in FIG. 3E; IV is CLEAVASE BBshown in FIG. 3F; V is the mutant shown in FIG. 4B; and VI is CLEAVASEBN shown in FIG. 3G.

Structure 2 was used to “normalize” the comparison. For example, it wasfound that it took 50 ng of Taq DNAP and 300 ng of CLEAVASE BN to givesimilar amounts of cleavage of Structure 2 in thirty (30) minutes. Underthese conditions native Taq DNAP is unable to cleave Structure 3 to anysignificant degree. Native Tfl DNAP cleaves Structure 3 in a manner thatcreates multiple products.

By contrast, all of the mutants tested cleave the linear duplex ofStructure 3. This finding indicates that this characteristic of themutant DNA polymerases is consistent of thermostable polymerases acrossthermophilic species.

Example 5 5′ Exonucleolytic Cleavage (“Nibbling”) by Thermostable DNAPs

It has been found that thermostable DNAPs, including those of thepresent invention, have a true 5′ exonuclease capable of nibbling the 5′end of a linear duplex nucleic acid structures. In this Example, the 206base pair DNA duplex substrate is again employed (See, Example 1C). Inthis case, it was produced by the use of one ³²P-labeled primer and oneunlabeled primer in a polymerase chain reaction. The cleavage reactionscomprised 0.01 pmoles of heat-denatured, end-labeled substrate DNA (withthe unlabeled strand also present), 5 pmoles of pilot oligonucleotide(see pilot oligos in FIG. 11A) and 0.5 units of DNAPTaq or 0.5 μ ofCLEAVASE BB in the E. coli extract (see above), in a total volume of 10μl of 10 mM Tris.Cl, pH 8.5, 50 mM KCl, 1.5 mM MgCl₂.

Reactions were initiated at 65° C. by the addition of pre-warmed enzyme,then shifted to the final incubation temperature for 30 minutes. Theresults are shown in FIG. 21A. Samples in lanes 1-4 are the results withnative Taq DNAP, while lanes 5-8 shown the results with CLEAVASE BB. Thereactions for lanes 1, 2, 5, and 6 were performed at 65° C. andreactions for lanes 3, 4, 7, and 8 were performed at 50° C. and all werestopped at temperature by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. Samples were heated to 75° C. for 2minutes immediately before electrophoresis through a 10% acrylamide gel(19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris.Borate, pH8.3, 1.4 mM EDTA. The expected product in reactions 1, 2, 5, and 6 is 85nucleotides long; in reactions 3 and 7, the expected product is 27nucleotides long. Reactions 4 and 8 were performed without pilot, andshould remain at 206 nucleotides. The faint band seen at 24 nucleotidesis residual end-labeled primer from the PCR.

The surprising result is that CLEAVASE BB under these conditions causesall of the label to appear in a very small species, suggesting thepossibility that the enzyme completely hydrolyzed the substrate. Todetermine the composition of the fastest-migrating band seen in lanes5-8 (reactions performed with the deletion mutant), samples of the 206base pair duplex were treated with either T7 gene 6 exonuclease (USB) orwith calf intestine alkaline phosphatase (Promega), according tomanufacturers' instructions, to produce either labeled mononucleotide(lane a of FIG. 21B) or free ³²P-labeled inorganic phosphate (lane b ofFIG. 21B), respectively. These products, along with the products seen inlane 7 of panel A were resolved by brief electrophoresis through a 20%acrylamide gel (19:1 cross-link), with 7 M urea, in a buffer of 45 mMTris.Borate, pH 8.3, 1.4 mM EDTA. CLEAVASE BB is thus capable ofconverting the substrate to mononucleotides.

Example 6 Nibbling is Duplex Dependent

The nibbling by CLEAVASE BB is duplex dependent. In this Example,internally labeled, single strands of the 206-mer were produced by 15cycles of primer extension incorporating α-³²P labeled dCTP combinedwith all four unlabeled dNTPs, using an unlabeled 206-bp fragment as atemplate. Single and double stranded products were resolved byelectrophoresis through a non-denaturing 6% polyacrylamide gel (29:1cross-link) in a buffer of 45 mM Tris.Borate, pH 8.3, 1.4 mM EDTA,visualized by autoradiography, excised from the gel, eluted by passivediffusion, and concentrated by ethanol precipitation.

The cleavage reactions comprised 0.04 pmoles of substrate DNA, and 2 μlof CLEAVASE BB (in an E. coli extract as described above) in a totalvolume of 40 μl of 10 mM Tris.Cl, pH 8.5, 50 mM KCl, 1.5 mM MgCl₂.Reactions were initiated by the addition of pre-warmed enzyme; 10 μlaliquots were removed at 5, 10, 20, and 30 minutes, and transferred toprepared tubes containing 8 μl of 95% formamide with 30 mM EDTA and0.05% marker dyes. Samples were heated to 75° C. for 2 minutesimmediately before electrophoresis through a 10% acrylamide gel (19:1cross-linked), with 7 M urea, in a buffer of 45 mM Tris.Borate, pH 8.3,1.4 mM EDTA. Results were visualized by autoradiography as shown in FIG.22. Clearly, the cleavage by CLEAVASE BB depends on a duplex structure;no cleavage of the single strand structure is detected whereas cleavageof the 206-mer duplex is complete.

Example 7 Nibbling can be Target Directed

The nibbling activity of the DNAPs of the present invention can beemployed with success in a detection assay. One embodiment of such anassay is shown in FIG. 23. In this assay, a labeled oligo is employedthat is specific for a target sequence. The oligo is in excess of thetarget so that hybridization is rapid. In this embodiment, the oligocontains two fluorescein labels whose proximity on the oligo causestheir emission to be quenched. When the DNAP is permitted to nibble theoligo the labels separate and are detectable. The shortened duplex isdestabilized and disassociates. Importantly, the target is now free toreact with an intact labeled oligo. The reaction can continue until thedesired level of detection is achieved. An analogous, althoughdifferent, type of cycling assay has been described employing lambdaexonuclease. See C. G. Copley and C. Boot, BioTechniques 13:888 (1992).

The success of such an assay depends on specificity. In other words, theoligo must hybridize to the specific target. It is also preferred thatthe assay be sensitive; the oligo ideally should be able to detect smallamounts of target. FIG. 24A shows a 5′ -end ³²P-labeled primer bound toa plasmid target sequence. In this case, the plasmid was pUC19(commercially available) which was heat denatured by boiling two (2)minutes and then quick chilling. The primer is a 21-mer (SEQ ID NO:28).The enzyme employed was CLEAVASE BX (a dilution equivalent to 5×10⁻³ μlextract) in 100 mM KCl, 10 mM Tris-Cl, pH 8.3, 2 mM MnCl₂. The reactionwas performed at 55° C. for sixteen (16) hours with or without genomicbackground DNA (from chicken blood). The reaction was stopped by theaddition of 8 μl of 95% formamide with 20 mM EDTA and marker dyes.

The products of the reaction were resolved by PAGE (10% polyacrylamide,19:1 cross link, 1× TBE) as seen in FIG. 24B. Lane “M” contains thelabeled 21-mer. Lanes 1-3 contain no specific target, although Lanes 2and 3 contain 100 ng and 200 ng of genomic DNA, respectively. Lanes 4, 5and 6 all contain specific target with either 0 ng, 100 ng, or 200 ng ofgenomic DNA, respectively. It is clear that conversion tomononucleotides occurs in Lanes 4, 5 and 6 regardless of the presence oramount of background DNA. Thus, the nibbling can be target directed andspecific.

Example 8 Cleavase Purification

As noted above, expressed thermostable proteins (i.e., the 5′nucleases), were isolated by crude bacterial cell extracts. Theprecipitated E. coli proteins were then, along with other cell debris,removed by centrifugation. In this Example, cells expressing the BNclone were cultured and collected (500 grams). For each gram (wetweight) of E. coli, 3 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mMEDTA, 100 μM NaCl) was added. The cells were lysed with 200 μg/mllysozyme at room temperature for 20 minutes. Thereafter deoxycholic acidwas added to make a 0.2% final concentration and the mixture wasincubated 15 minutes at room temperature.

The lysate was sonicated for approximately 6-8 minutes at 0° C. Theprecipitate was removed by centrifugation (39,000 g for 20 minutes).Polyethyleneimine was added (0.5%) to the supernatant and the mixturewas incubated on ice for 15 minutes. The mixture was centrifuged (5,000g for 15 minutes) and the supernatant was retained. This was heated for30 minutes at 60° C. and then centrifuged again (5,000 g for 15 minutes)and the supernatant was again retained.

The supernatant was precipitated with 35% ammonium sulfate at 4° C. for15 minutes. The mixture was then centrifuged (5,000 g for 15 minutes)and the supernatant was removed. The precipitate was then dissolved in0.25M KCl, 20 Tris pH 7.6, 0.2% Tween and 0.1 EDTA) and then dialyzedagainst Binding Buffer (8× Binding Buffer comprises: 40 mM imidazole, 4MNaCl, 160 mM Tris-HCl, pH 7.9).

The solubilized protein is then purified on the Ni⁺⁺ column (Novagen).The Binding Buffer is allows to drain to the top of the column bed andload the column with the prepared extract. A flow rate of about 10column volumes per hour is optimal for efficient purification. If theflow rate is too fast, more impurities will contaminate the elutedfraction.

The column is washed with 25 ml (10 volumes) of 1× Binding Buffer andthen washed with 15 ml (6 volumes) of 1× Wash Buffer (8× Wash Buffercomprises: 480 mM imidazole, 4 M NaCl, 160 mM Tris-HCl, pH 7.9). Thebound protein was eluted with 15 ml (6 volumes) of 1× Elute Buffer (4×Elute Buffer comprises: 4mM imidazole, 2 M NaCl, 80 mM Tris-HCl, pH7.9). Protein is then reprecipitated with 35% ammonium sulfate as above.The precipitate was then dissolved and dialyzed against: 20 mM Tris, 100mM KCl, 1 mM EDTA). The solution was brought up to 0.1 % each of Tween20 and NP-40 and stored at 4° C.

Example 9 The Use of Various Divalent Cations in the Cleavage ReactionInfluences the Nature of the Resulting Cleavage Products

In comparing the 5′ nucleases generated by the modification and/ordeletion of the C-terminal polymerization domain of Thermus aquaticusDNA polymerase (DNAPTaq), as diagrammed in FIG. 3B-G, significantdifferences in the strength of the interactions of these proteins withthe 3′ end of primers located upstream of the cleavage site (as depictedin FIG. 5) were noted. In describing the cleavage of these structures byPol I-type DNA polymerases (See, Example 1, and Lyamichev et al.,Science 260:778 [1993]), it was observed that in the absence of aprimer, the location of the junction between the double-stranded regionand the single-stranded 5′ and 3′ arms determined the site of cleavage,but in the presence of a primer, the location of the 3′ end of theprimer became the determining factor for the site of cleavage. It waspostulated that this affinity for the 3′ end was in accord with thesynthesizing function of the DNA polymerase.

Structure 2, shown in FIG. 20A, was used to test the effects of a 3′ endproximal to the cleavage site in cleavage reactions comprising severaldifferent solutions (e.g., solutions containing different salts [KCl orNaCl], different divalent cations [Mn²⁺ or Mg²⁺], etc.) as well as theuse of different temperatures for the cleavage reaction. When thereaction conditions were such that the binding of the enzyme (e.g., aDNAP comprising a 5′ nuclease, a modified DNAP or a 5′ nuclease) to the3′ end (of the pilot oligonucleotide) near the cleavage site was strong,the structure shown is cleaved at the site indicated in FIG. 20A. Thiscleavage releases the unpaired 5′ arm and leaves a nick between theremaining portion of the target nucleic acid and the folded 3′ end ofthe pilot oligonucleotide. In contrast, when the reaction conditions aresuch that the binding of the DNAP (comprising a 5′ nuclease) to the 3′end was weak, the initial cleavage was as described above, but after therelease of the 5′ arm, the remaining duplex is digested by theexonuclease function of the DNAP.

One way of weakening the binding of the DNAP to the 3′ end is to removeall or part of the domain to which at least some of this function hasbeen attributed. Some of 5′ nucleases created by deletion of thepolymerization domain of DNAPTaq have enhanced true exonucleasefunction, as demonstrated in Example 5.

The affinity of these types of enzymes (i.e., 5′ nucleases associatedwith or derived from DNAPs) for recessed 3′ ends may also be affected bythe identity of the divalent cation present in the cleavage reaction. Itwas demonstrated by Longley et al. (Nucl. Acids Res., 18:7317 [1990])that the use of MnCl₂ in a reaction with DNAPTaq enabled the polymeraseto remove nucleotides from the 5′ end of a primer annealed to atemplate, albeit inefficiently. Similarly, by examination of thecleavage products generated using Structure 2 from FIG. 20A, asdescribed above, in a reaction containing either DNAPTaq or the CLEAVASEBB nuclease, it was observed that the substitution of MnCl₂ for MgCl₂ inthe cleavage reaction resulted in the exonucleolytic “nibbling” of theduplex downstream of the initial cleavage site. While not limiting theinvention to any particular mechanism, it is thought that thesubstitution of MnCl₂ for MgCl₂ in the cleavage reaction lessens theaffinity of these enzymes for recessed 3′ ends.

In all cases, the use of MnCl₂ enhances the 5′ nuclease function, and inthe case of the CLEAVASE BB nuclease, a 50-to 100-fold stimulation ofthe 5′ nuclease function is seen. Thus, while the exonuclease activityof these enzymes was demonstrated above in the presence of MgCl₂, theassays described below show a comparable amount of exonuclease activityusing 50 to 100-fold less enzyme when MnCl₂ is used in place of MgCl₂.When these reduced amounts of enzyme are used in a reaction mixturecontaining MgCl₂, the nibbling or exonuclease activity is much lessapparent than that seen in Examples 5-7.

Similar effects are observed in the performance of the nucleic aciddetection assay described in Examples 10-39 below when reactionsperformed in the presence of either MgCl₂ or MnCl₂ are compared. In thepresence of either divalent cation, the presence of the INVADERoligonucleotide (described below) forces the site of cleavage into theprobe duplex, but in the presence of MnCl₂ the probe duplex can befurther nibbled producing a ladder of products that are visible when a3′ end label is present on the probe oligonucleotide. When the INVADERoligonucleotide is omitted from a reaction containing Mn²+, the probe isnibbled from the 5′ end. Mg²+-based reactions display minimal nibblingof the probe oligonucleotide. In any of these cases, the digestion ofthe probe is dependent upon the presence of the target nucleic acid. Inthe examples below, the ladder produced by the enhanced nibblingactivity observed in the presence of Mn²⁺ is used as a positiveindicator that the probe oligonucleotide has hybridized to the targetsequence.

Example 10 Invasive 5′ Endonucleolytic Cleavage by Thermostable 5′Nucleases in the Absence of Polymerization

As described in the Examples above, 5′ nucleases cleave near thejunction between single-stranded and base-paired regions in a bifurcatedduplex, usually about one base pair into the base-paired region. In thisExample, it is shown that thermostable 5′ nucleases, including those ofthe present invention (e.g., CLEAVASE BN nuclease, CLEAVASE A/Gnuclease), have the ability to cleave a greater distance into the basepaired region when provided with an upstream oligonucleotide bearing a3′ region that is homologous to a 5′ region of the subject duplex, asshown in FIG. 26.

FIG. 26 shows a synthetic oligonucleotide that was designed to fold uponitself and that consists of the following sequence:5′-GTTCTCTGCTCTCTGGTCGCTG TCTCGCTTGTGAAACAAGCGAGACAGCGTGGTCTCTCG-3′ (SEQID NO:29). This oligonucleotide is referred to as the “S-60 Hairpin.”The 15 basepair hairpin formed by this oligonucleotide is furtherstabilized by a “tri-loop” sequence in the loop end (i.e., threenucleotides form the loop portion of the hairpin) (Hiraro et al.,Nucleic Acids Res., 22(4):576 [1994]). FIG. 26 also show the sequence ofthe P-15 oligonucleotide and the location of the region ofcomplementarity shared by the P-15 and S-60 hairpin oligonucleotides.The sequence of the P-15 oligonucleotide is 5′-CGAGAGACCACGCTG-3′ (SEQID NO:30). As discussed in detail below, the solid black arrowheadsshown in FIG. 26 indicate the sites of cleavage of the S-60 hairpin inthe absence of the P-15 oligonucleotide and the hollow arrow headsindicate the sites of cleavage in the presence of the P-15oligonucleotide. The size of the arrow head indicates the relativeutilization of a particular site.

The S-60 hairpin molecule was labeled on its 5′ end with biotin forsubsequent detection. The S-60 hairpin was incubated in the presence ofa thermostable 5′ nuclease in the presence or the absence of the P-15oligonucleotide. The presence of the full duplex that can be formed bythe S-60 hairpin is demonstrated by cleavage with the CLEAVASE BN 5′nuclease, in a primer-independent fashion (i.e., in the absence of theP-15 oligonucleotide). The release of 18 and 19-nucleotide fragmentsfrom the 5′ end of the S-60 hairpin molecule showed that the cleavageoccurred near the junction between the single and double strandedregions when nothing is hybridized to the 3′ arm of the S-60 hairpin(FIG. 27, lane 2).

The reactions shown in FIG. 27 were conducted as follows. Twenty fmoleof the 5′ biotin-labeled hairpin DNA (SEQ ID NO:29) was combined with0.1 ng of CLEAVASE BN enzyme and 1 μl of 100 mM MOPS (pH 7.5) containing0.5% each of Tween-20 and NP-40 in a total volume of 9 μl. In thereaction shown in lane 1, the enzyme was omitted and the volume was madeup by addition of distilled water (this served as the uncut or no enzymecontrol). The reaction shown in lane 3 of FIG. 27 also included 0.5pmole of the P15 oligonucleotide (SEQ ID NO:30), which can hybridize tothe unpaired 3′ arm of the S-60 hairpin (SEQ ID NO:29), as diagrammed inFIG. 26.

The reactions were overlaid with a drop of mineral oil, heated to 95° C.for 15 seconds, then cooled to 37° C., and the reaction was started bythe addition of 1 μl of 10 mM MnCl₂ to each tube. After 5 minutes, thereactions were stopped by the addition of 6 μl of 95% formamidecontaining 20 mM EDTA and 0.05% marker dyes. Samples were heated to 75°C. for 2 minutes immediately before electrophoresis through a 15%acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated allowing the gel toremain flat on one plate. A 0.2 mm-pore positively-charged nylonmembrane (NYTRAN, Schleicher and Schuell, Keene, N.H.), pre-wetted inH₂O, was laid on top of the exposed gel. All air bubbles were removed.Two pieces of 3 MM filter paper (Whatman) were then placed on top of themembrane, the other glass plate was replaced, and the sandwich wasclamped with binder clips. Transfer was allowed to proceed overnight.After transfer, the membrane was carefully peeled from the gel andallowed to air dry. After complete drying, the membrane was washed in1.2× Sequenase Images Blocking Buffer (United States Biochemical) using0.3 ml of buffer/cm of membrane. The wash was performed for 30 minutesat room temperature. A streptavidin-alkaline phosphatase conjugate(SAAP, United States Biochemical) was added to a 1:4000 dilutiondirectly to the blocking solution, and agitated for 15 minutes. Themembrane was rinsed briefly with H₂O and then washed three times for 5minutes per wash using 0.5 ml/cm² of 1× SAAP buffer (100 mM Tris-HCl, pH10, 50 mM NaCl) with 0.1% sodium dodecyl sulfate (SDS). The membrane wasrinsed briefly with H₂O between each wash. The membrane was then washedonce in 1× SAAP buffer containing 1 mM MgCl₂ without SDS, drainedthoroughly and placed in a plastic heat-sealable bag. Using a sterilepipet, 5 mls of CDP-Star™ (Tropix, Bedford, Mass.) chemiluminescentsubstrate for alkaline phosphatase were added to the bag and distributedover the entire membrane for 2-3 minutes. The CDP-Star™-treated membranewas exposed to XRP X-ray film (Kodak) for an initial exposure of 10minutes.

The resulting autoradiograph is shown in FIG. 27. In FIG. 27, the lanelabeled “M” contains the biotinylated P-15 oligonucleotide, which servedas a marker. The sizes (in nucleotides) of the uncleaved S-60 hairpin(60 nuc; lane 1), the marker (15 nuc; lane “M”) and the cleavageproducts generated by cleavage of the S-60 hairpin in the presence (lane3) or absence (lane 2) of the P-15 oligonucleotide are indicated.

Because the complementary regions of the S-60 hairpin are located on thesame molecule, essentially no lag time should be needed to allowhybridization (i.e., to form the duplex region of the hairpin). Thishairpin structure would be expected to form long before the enzyme couldlocate and cleave the molecule. As expected, cleavage in the absence ofthe primer oligonucleotide was at or near the junction between theduplex and single-stranded regions, releasing the unpaired 5′ arm (FIG.27, lane 2). The resulting cleavage products were 18 and 19 nucleotidesin length.

It was expected that stability of the S-60 hairpin with the tri-loopwould prevent the P-15 oligonucleotide from promoting cleavage in the“primer-directed” manner described in Example 1 above, because the 3′end of the “primer” would remain unpaired. Surprisingly, it was foundthat the enzyme seemed to mediate an “invasion” by the P-15 primer intothe duplex region of the S-60 hairpin, as evidenced by the shifting ofthe cleavage site 3 to 4 basepairs further into the duplex region,releasing the larger products (22 and 21 nuc.) observed in lane 3 ofFIG. 27.

The precise sites of cleavage of the S-60 hairpin are diagrammed on thestructure in FIG. 26, with the solid black arrowheads indicating thesites of cleavage in the absence of the P-15 oligonucleotide and thehollow arrow heads indicating the sites of cleavage in the presence ofP-15.

These data show that the presence on the 3′ arm of an oligonucleotidehaving some sequence homology with the first several bases of thesimilarly oriented strand of the downstream duplex can be a dominantfactor in determining the site of cleavage by 5′ nucleases. Because theoligonucleotide that shares some sequence homology with the firstseveral bases of the similarly oriented strand of the downstream duplexappears to invade the duplex region of the hairpin, it is referred to asan “INVADER” oligonucleotide. As shown in the Examples below, an INVADERoligonucleotide appears to invade (or displace) a region of duplexednucleic acid regardless of whether the duplex region is present on thesame molecule (i.e., a hairpin) or whether the duplex is formed betweentwo separate nucleic acid strands.

Example 11 The INVADER Oligonucleotide Shifts the Site of Cleavage in aPre-Formed Probe/Target Duplex

In Example 10, it was demonstrated that an INVADER oligonucleotide couldshift the site at which a 5′ nuclease cleaves a duplex region present ona hairpin molecule. In this Example, the ability of an INVADERoligonucleotide to shift the site of cleavage within a duplex regionformed between two separate strands of nucleic acid molecules wasexamined.

A single-stranded target DNA comprising the single-stranded circularM13mp19 molecule and a labeled (fluorescein) probe oligonucleotide weremixed in the presence of the reaction buffer containing salt (KCl) anddivalent cations (Mg²⁺ or Mn²⁺) to promote duplex formation. The probeoligonucleotide refers to a labeled oligonucleotide that iscomplementary to a region along the target molecule (e.g., M13mp19). Asecond oligonucleotide (unlabeled) was added to the reaction after theprobe and target had been allowed to anneal. The second oligonucleotidebinds to a region of the target that is located downstream of the regionto which the probe oligonucleotide binds. This second oligonucleotidecontains sequences that are complementary to a second region of thetarget molecule. If the second oligonucleotide contains a region that iscomplementary to a portion of the sequences along the target to whichthe probe oligonucleotide also binds, this second oligonucleotide isreferred to as an INVADER oligonucleotide (see FIG. 28c).

FIG. 32 depicts the annealing of two oligonucleotides to regions alongthe M13mp19 target molecule (bottom strand in all three structuresshown). In FIG. 28 only a 52 nucleotide portion of the M13mp19 moleculeis shown; this 52 nucleotide sequence is listed in SEQ ID NO:31. Theprobe oligonucleotide contains a fluorescein label at the 3′ end; thesequence of the probe is 5′-AGAAAGGAAGGGAAGAAAGCGAAAGG-3′ (SEQ IDNO:32). In FIG. 28, sequences comprising the second oligonucleotide,including the INVADER oligonucleotide are underlined. In FIG. 28 a, thesecond oligonucleotide, which has the sequence 5′-GACGGGGAAAGCCGGCGAACG-3′ (SEQ ID NO:33), is complementary to adifferent and downstream region of the target molecule than is the probeoligonucleotide (labeled with fluorescein or “Fluor”); there is a gapbetween the second, upstream oligonucleotide and the probe for thestructure shown in FIG. 28 a. In FIG. 28 b, the second, upstreamoligonucleotide, which has the sequence 5′-GAAAGCCGGCGAACGTGGCG-3′ (SEQID NO:34), is complementary to a different region of the target moleculethan is the probe oligonucleotide, but in this case, the secondoligonucleotide and the probe oligonucleotide abut one another (that isthe 3′ end of the second, upstream oligonucleotide is immediatelyadjacent to the 5′ end of the probe such that no gap exists betweenthese two oligonucleotides). In FIG. 28 c, the second, upstreamoligonucleotide (5′-GGCGAACGTGGCGAGAAAGGA-3′ [SEQ ID NO:35]) and theprobe oligonucleotide share a region of complementarity with the targetmolecule. Thus, the upstream oligonucleotide has a 3′ arm that has asequence identical to the first several bases of the downstream probe.In this situation, the upstream oligonucleotide is referred to as an“INVADER” oligonucleotide.

The effect of the presence of an INVADER oligonucleotide upon thepattern of cleavage in a probe/target duplex formed prior to theaddition of the INVADER was examined. The INVADER oligonucleotide andthe enzyme were added after the probe was allowed to anneal to thetarget and the position and extent of cleavage of the probe wereexamined to determine a) if the INVADER was able to shift the cleavagesite to a specific internal region of the probe, and b), if the reactioncould accumulate specific cleavage products over time, even in theabsence of thermal cycling, polymerization, or exonuclease removal ofthe probe sequence.

The reactions were carried out as follows. Twenty μl each of two enzymemixtures were prepared, containing 2 μl of CLEAVASE A/G nuclease extract(prepared as described in Example 2), with or without 50 pmole of theINVADER oligonucleotide (SEQ ID NO:35), as indicated, per 4 μl of themixture. For each of the eight reactions shown in FIG. 29, 150 fmole ofM13mp19 single-stranded DNA (available from Life Technologies, Inc.) wascombined with 5 pmoles of fluorescein labeled probe (SEQ ID NO:32), tocreate the structure shown in FIG. 28 c, but without the INVADERoligonucleotide present (the probe/target mixture). One half (4 tubes)of the probe/target mixtures were combined with 1 μl of 100 mM MOPS, pH7.5 with 0.5% each of Tween-20 and NP-40, 0.5 μl of 1 M KCl and 0.25 μlof 80 mM MnCl₂, and distilled water to a volume of 6 μl. The second setof probe/target mixtures were combined with 1 μl of 100 mM MOPS, pH 7.5with 0.5% each of Tween-20 and NP-40, 0.5 μl of 1 M KCl and 0.25 μl of80 mM MgCl₂. The second set of mixtures therefore contained MgCl₂ inplace of the MnCl₂ present in the first set of mixtures.

The mixtures (containing the probe/target with buffer, KCl and divalentcation) were covered with a drop of CHILLOUT evaporation barrier andwere brought to 60° C. for 5 minutes to allow annealing. Four μl of theabove enzyme mixtures without the INVADER oligonucleotide was added toreactions whose products are shown in lanes 1, 3, 5 and 7 of FIG. 29.Reactions whose products are shown lanes 2, 4, 6, and 8 of FIG. 29received the same amount of enzyme mixed with the INVADERoligonucleotide (SEQ ID NO:35). Reactions 1, 2, 5 and 6 were incubatedfor 5 minutes at 60° C. and reactions 3, 4, 7 and 8 were incubated for15 minutes at 60° C.

All reactions were stopped by the addition of 8 μl of 95% formamide with20 mM EDTA and 0.05% marker dyes. Samples were heated to 90° C. for 1minute immediately before electrophoresis through a 20% acrylamide gel(19:1 cross-linked), containing 7 M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA. Following electrophoresis, thereaction products and were visualized by the use of an Hitachi FMBIOfluorescence imager, the output of which is seen in FIG. 29. The verylow molecular weight fluorescent material seen in all lanes at or nearthe salt front in FIG. 29 and other fluoro-imager Figures is observedwhen fluorescently-labeled oligonucleotides are electrophoresed andimaged on a fluoro-imager. This material is not a product of thecleavage reaction.

The use of MnCl₂ in these reactions (lanes 1-4) stimulates the trueexonuclease or “nibbling” activity of the CLEAVASE enzyme, as describedin Example 6, as is clearly seen in lanes 1 and 3 of FIG. 29. Thisnibbling of the probe oligonucleotide (SEQ ID NO:32) in the absence ofINVADER oligonucleotide (SEQ ID NO:35) confirms that the probeoligonucleotide is forming a duplex with the target sequence. Theladder-like products produced by this nibbling reaction may be difficultto differentiate from degradation of the probe by nucleases that mightbe present in a clinical specimen. In contrast, introduction of theINVADER oligonucleotide (SEQ ID NO:35) caused a distinctive shift in thecleavage of the probe, pushing the site of cleavage 6 to 7 bases intothe probe, confirming the annealing of both oligonucleotides. Inpresence of MnCl₂, the exonuclease “nibbling” may occur after theINVADER-directed cleavage event, until the residual duplex isdestabilized and falls apart.

In a magnesium based cleavage reaction (lanes 5-8), the nibbling or trueexonuclease function of the CLEAVASE A/G is enzyme suppressed (but theendonucleolytic function of the enzyme is essentially unaltered), so theprobe oligonucleotide is not degraded in the absence of the INVADER(FIG. 29, lanes 5 and 7). When the INVADER is added, it is clear thatthe INVADER oligonucleotide can promote a shift in the site of theendonucleolytic cleavage of the annealed probe. Comparison of theproducts of the 5 and 15 minute reactions with INVADER (lanes 6 and 8 inFIG. 29) shows that additional probe hybridizes to the target and iscleaved. The calculated melting temperature (T_(m)) of the portion ofprobe that is not invaded (i.e., nucleotides 9-26 of SEQ ID NO:32) is56° C., so the observed turnover (as evidenced by the accumulation ofcleavage products with increasing reaction time) suggests that the fulllength of the probe molecule, with a calculated T_(m) of 76° C., is mustbe involved in the subsequent probe annealing events in this 60° C.reaction.

Example 12 The Overlap of the 3′ INVADER Oligonucleotide Sequence withthe 5′ Region of the Probe Causes a Shift in the Site of Cleavage

In Example 11, the ability of an INVADER oligonucleotide to cause ashift in the site of cleavage of a probe annealed to a target moleculewas demonstrated. In this Example, experiments were conducted to examinewhether the presence of an oligonucleotide upstream from the probe wassufficient to cause a shift in the cleavage site(s) along the probe orwhether the presence of nucleotides on the 3′ end of the INVADERoligonucleotide that have the same sequence as the first severalnucleotides at the 5′ end of the probe oligonucleotide were required topromote the shift in cleavage.

To examine this point, the products of cleavage obtained from threedifferent arrangements of target-specific oligonucleotides are compared.A diagram of these oligonucleotides and the way in which they hybridizeto a test nucleic acid, M13mp19, is shown in FIG. 28. In FIG. 28 a, the3′ end of the upstream oligonucleotide (SEQ ID NO:33) is locatedupstream of the 5′ end of the downstream “probe” oligonucleotide (SEQ IDNO:32) such that a region of the M13 target that is not paired to eitheroligonucleotide is present. In FIG. 28 b, the sequence of the upstreamoligonucleotide (SEQ ID NO:34) is immediately upstream of the probe (SEQID NO:32), having neither a gap nor an overlap between the sequences.FIG. 28 c diagrams the arrangement of the substrates used in the assayof the present invention, showing that the upstream “INVADER”oligonucleotide (SEQ ID NO:35) has the same sequence on a portion of its3′ region as that present in the 5′ region of the downstream probe (SEQID NO:32). That is to say, these regions will compete to hybridize tothe same segment of the M13 target nucleic acid.

In these experiments, four enzyme mixtures were prepared as follows(planning 5 μl per digest): Mixture 1 contained 2.25 μl of CLEAVASE A/Gnuclease extract (prepared as described in Example 2) per 5 μl ofmixture, in 20 mM MOPS, pH 7.5 with 0.1% each of Tween 20 and NP-40, 4mM MnCl₂ and 100 mM KCl. Mixture 2 contained 11.25 units of Taq DNApolymerase (Promega) per 5 μl of mixture in 20 mM MOPS, pH 7.5 with 0.1%each of Tween 20 and NP-40, 4 mM MnCl₂ and 100 mM KCl. Mixture 3contained 2.25 μl of CLEAVASE A/G nuclease extract per 5 μl of mixturein 20 mM Tris-HCl, pH 8.5, 4 mM MgCl₂ and 100 mM KCl. Mixture 4contained 11.25 units of Taq DNA polymerase per 5 μl of mixture in 20 mMTris-HCl, pH 8.5, 4 mM MgCl₂ and 100 mM KCl.

For each reaction, 50 fmole of M13mp19 single-stranded DNA (the targetnucleic acid) was combined with 5 pmole of the probe oligonucleotide(SEQ ID NO:32 which contained a fluorescein label at the 3′ end) and 50pmole of one of the three upstream oligonucleotides diagrammed in FIG.28 (i.e., one of SEQ ID NOS:33-35), in a total volume of 5 μl ofdistilled water. The reactions were overlaid with a drop of ChillOut™evaporation barrier and warmed to 62° C. The cleavage reactions werestarted by the addition of 5 μl of an enzyme mixture to each tube, andthe reactions were incubated at 62° C. for 30 min. The reactions shownin lanes 1-3 of FIG. 30 received Mixture 1; reactions 4-6 receivedMixture 2; reactions 7-9 received Mixture 3 and reactions 10-12 receivedMixture 4.

After 30 minutes at 62° C., the reactions were stopped by the additionof 8 μl of 95% formamide with 20 mM EDTA and 0.05% marker dyes. Sampleswere heated to 75° C. for 2 minutes immediately before electrophoresisthrough a 20% acrylamide gel (19:1 cross-linked), with 7 M urea, in abuffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

Following electrophoresis, the products of the reactions were visualizedby the use of an Hitachi FMBIO fluorescence imager, the output of whichis seen in FIG. 30. The reaction products shown in lanes 1, 4, 7 and 10of FIG. 30 were from reactions that contained SEQ ID NO:33 as theupstream oligonucleotide (see FIG. 28 a). The reaction products shown inlanes 2, 5, 8 and 11 of FIG. 30 were from reactions that contained SEQID NO:34 as the upstream oligonucleotide (see FIG. 28 b). The reactionproducts shown in lanes 3, 6, 9 and 12 of FIG. 30 were from reactionsthat contained SEQ ID NO:35, the INVADER oligonucleotide, as theupstream oligonucleotide (see FIG. 28 c).

Examination of the Mn²⁺ based reactions using either CLEAVASE A/Gnuclease or DNAPTaq as the cleavage agent (lanes 1 through 3 and 4through 6, respectively) shows that both enzymes have active exonucleasefunction in these buffer conditions. The use of a 3′ label on the probeoligonucleotide allows the products of the nibbling activity to remainlabeled, and therefore visible in this assay. The ladders seen in lanes1, 2, 4 and 5 confirm that the probe hybridize to the target DNA asintended. These lanes also show that the location of the non-invasiveoligonucleotides have little effect on the products generated. Theuniform ladder created by these digests would be difficult todistinguish from a ladder causes by a contaminating nuclease, as onemight find in a clinical specimen. In contrast, the products displayedin lanes 3 and 6, where an INVADER oligonucleotide was provided todirect the cleavage, show a very distinctive shift, so that the primarycleavage product is smaller than those seen in the non-invasivecleavage. This product is then subject to further nibbling in theseconditions, as indicated by the shorter products in these lanes. TheseINVADER-directed cleavage products would be easily distinguished from abackground of non-specific degradation of the probe oligonucleotide.

When Mg²⁺ is used as the divalent cation the results are even moredistinctive. In lanes 7, 8, 10 and 11 of FIG. 30, where the upstreamoligonucleotides were not invasive, minimal nibbling is observed. Theproducts in the DNAPTaq reactions show some accumulation of probe thathas been shortened on the 5′ end by one or two nucleotides consistentwith previous examination of the action of this enzyme on nickedsubstrates (Longley et al., supra). When the upstream oligonucleotide isinvasive, however, the appearance of the distinctively shifted probeband is seen. These data clearly indicated that it is the invasive 3′portion of the upstream oligonucleotide that is responsible for fixingthe site of cleavage of the downstream probe.

Thus, the above results demonstrate that it is the presence of the freeor initially non-annealed nucleotides at the 3′ end of the INVADERoligonucleotide that mediate the shift in the cleavage site, not justthe presence of an oligonucleotide annealed upstream of the probe.Nucleic acid detection assays that employ the use of an INVADERoligonucleotide are termed “INVADER-directed cleavage” assays.

Example 13 INVADER-Directed Cleavage Recognizes Single and DoubleStranded Target Molecules in a Background of Non-Target DNA Molecules

For a nucleic acid detection method to be broadly useful, it must beable to detect a specific target in a sample that may contain largeamounts of other DNA, (e.g., bacterial or human chromosomal DNA). Theability of the INVADER directed cleavage assay to recognize and cleaveeither single- or double-stranded target molecules in the presence oflarge amounts of non-target. DNA was examined. In these experiments amodel target nucleic acid, M13, in either single or double stranded form(single-stranded M13mp18 is available from Life Technologies, Inc anddouble-stranded M13mp19 is available from NEB), was combined with humangenomic DNA (Novagen) and then utilized in INVADER-directed cleavagereactions. Before the start of the cleavage reaction, the DNAs wereheated to 95° C. for 15 minutes to completely denature the samples, asis standard practice in assays, such as polymerase chain reaction orenzymatic DNA sequencing, which involve solution hybridization ofoligonucleotides to double-stranded target molecules.

For each of the reactions shown in lanes 2-5 of FIG. 31, the target DNA(25 fmole of the ss DNA or 1 pmole of the ds DNA) was combined with 50pmole of the INVADER oligonucleotide (SEQ ID NO:35); for the reactionshown in lane 1 the target DNA was omitted. Reactions 1, 3 and 5 alsocontained 470 ng of human genomic DNA. These mixtures were brought to avolume of 10 μl with distilled water, overlaid with a drop of ChillOut™evaporation barrier, and brought to 95° C. for 15 minutes. After thisincubation period, and still at 95° C., each tube received 10 μl of amixture comprising 2.25 μl of CLEAVASE A/G nuclease extract (prepared asdescribed in Example 2) and 5 pmole of the probe oligonucleotide (SEQ IDNO:32), in 20 mM MOPS, pH 7.5 with 0.1% each of Tween 20 and NP-40, 4 mMMnCl₂ and 100 mM KCl. The reactions were brought to 62° C. for 15minutes and stopped by the addition of 12 μl of 95% formamide with 20 mMEDTA and 0.05% marker dyes. Samples were heated to 75° C. for 2 minutesimmediately before electrophoresis through a 20% acrylamide gel (19:1cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3,1.4 mM EDTA. The products of the reactions were visualized by the use ofan Hitachi FMBIO fluorescence imager. The results are displayed in FIG.31.

In FIG. 31, lane 1 contains the products of the reaction containing theprobe (SEQ ID NO:32), the INVADER oligonucleotide (SEQ ID NO:35) andhuman genomic DNA. Examination of lane 1 shows that the probe andINVADER oligonucleotides are specific for the target sequence, and thatthe presence of genomic DNA does not cause any significant backgroundcleavage.

In FIG. 31, lanes 2 and 3 contain reaction products from reactionscontaining the single-stranded target DNA (M13mp18), the probe (SEQ IDNO:32) and the INVADER oligonucleotide (SEQ ID NO:35) in the absence orpresence of human genomic DNA, respectively. Examination of lanes 2 and3 demonstrate that the INVADER detection assay may be used to detect thepresence of a specific sequence on a single-stranded target molecule inthe presence or absence of a large excess of competitor DNA (humangenomic DNA).

In FIG. 31, lanes 4 and 5 contain reaction products from reactionscontaining the double-stranded target DNA (M13mp19), the probe (SEQ IDNO:32) and the INVADER oligonucleotide (SEQ ID NO:35) in the absence orpresence of human genomic DNA, respectively. Examination of lanes 4 and5 show that double stranded target molecules are eminently suitable forINVADER-directed detection reactions. The success of this reaction usinga short duplexed molecule, M13mp19, as the target in a background of alarge excess of genomic DNA is especially noteworthy as it would beanticipated that the shorter and less complex M13 DNA strands would beexpected to find their complementary strand more easily than would thestrands of the more complex human genomic DNA. If the M13 DNA reannealedbefore the probe and/or INVADER oligonucleotides could bind to thetarget sequences along the M13 DNA, the cleavage reaction would beprevented. In addition, because the denatured genomic DNA wouldpotentially contain regions complementary to the probe and/or INVADERoligonucleotides it was possible that the presence of the genomic DNAwould inhibit the reaction by binding these oligonucleotides therebypreventing their hybridization to the M13 target. The above resultsdemonstrate that these theoretical concerns are not a problem under thereaction conditions employed above.

In addition to demonstrating that the INVADER detection assay may beused to detect sequences present in a double-stranded target, these dataalso show that the presence of a large amount of non-target DNA (470ng/20 μl reaction) does not lessen the specificity of the cleavage.While this amount of DNA does show some impact on the rate of productaccumulation, probably by binding a portion of the enzyme, the nature ofthe target sequence, whether single- or double-stranded nucleic acid,does not limit the application of this assay.

Example 14 Signal Accumulation in the INVADER-Directed Cleavage Assay asa Function of Target Concentration

To investigate whether the INVADER-directed cleavage assay could be usedto indicate the amount of target nucleic acid in a sample, the followingexperiment was performed. Cleavage reactions were assembled thatcontained an INVADER oligonucleotide (SEQ ID NO:35), a labeled probe(SEQ ID NO:32) and a target nucleic acid, M13mp19. A series ofreactions, which contained smaller and smaller amounts of the M13 targetDNA, was employed in order to examine whether the cleavage productswould accumulate in a manner that reflected the amount of target DNApresent in the reaction.

The reactions were conducted as follows. A master mix containing enzymeand buffer was assembled. Each 5 μl of the master mixture contained 25ng of CLEAVASE BN nuclease in 20 mM MOPS (pH 7.5) with 0.1% each ofTween 20 and NP-40, 4 mM MnCl₂ and 100 mM KCl. For each of the cleavagereactions shown in lanes 4-13 of FIG. 32, a DNA mixture was generatedthat contained 5 pmoles of the fluorescein-labeled probe oligonucleotide(SEQ ID NO:32), 50 pmoles of the INVADER oligonucleotide (SEQ ID NO:35)and 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01 or 0.005 fmoles ofsingle-stranded M13mp19, respectively, for every 5 μl of the DNAmixture. The DNA solutions were covered with a drop of CHILLOUTevaporation barrier and brought to 61° C. The cleavage reactions werestarted by the addition of 5 μl of the enzyme mixture to each of tubes(final reaction volume was 10 μl). After 30 minutes at 61° C., thereactions were terminated by the addition of 8 μl of 95% formamide with20 mM EDTA and 0.05% marker dyes. Samples were heated to 90° C. for 1minute immediately before electrophoresis through a 20% denaturingacrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer containing45 mM Tris-Borate (pH 8.3), 1.4 mM EDTA. To provide reference (i.e.,standards), 1.0, 0.1 and 0.01 pmole aliquots of fluorescein-labeledprobe oligonucleotide (SEQ ID NO:32) were diluted with the aboveformamide solution to a final volume of 18 μl . These reference markerswere loaded into lanes 1-3, respectively of the gel. The products of thecleavage reactions (as well as the reference standards) were visualizedfollowing electrophoresis by the use of a Hitachi FMBIO fluorescenceimager. The results are displayed in FIG. 32.

In FIG. 32, boxes appear around fluorescein-containing nucleic acid(i.e., the cleaved and uncleaved probe molecules) and the amount offluorescein contained within each box is indicated under the box. Thebackground fluorescence of the gel (see box labeled “background”) wassubtracted by the fluoro-imager to generate each value displayed under abox containing cleaved or uncleaved probe products (the boxes arenumbered 1-14 at top left with a V followed by a number below the box).The lane marked “M” contains fluoresceinated oligonucleotides, whichserved as markers.

The results shown in FIG. 32, demonstrate that the accumulation ofcleaved probe molecules in a fixed-length incubation period reflects theamount of target DNA present in the reaction. The results alsodemonstrate that the cleaved probe products accumulate in excess of thecopy number of the target. This is clearly demonstrated by comparing theresults shown in lane 3, in which 10 fmole (0.01 pmole) of uncut probeare displayed with the results shown in 5, where the products thataccumulated in response to the presence of 10 fmole of target DNA aredisplayed. These results show that the reaction can cleave hundreds ofprobe oligonucleotide molecules for each target molecule present,dramatically amplifying the target-specific signal generated in theINVADER-directed cleavage reaction.

Example 15 Effect of Saliva Extract on the INVADER-Directed CleavageAssay

For a nucleic acid detection method to be useful in a medical (i.e., adiagnostic) setting, it must not be inhibited by materials andcontaminants likely to be found in a typical clinical specimen. To testthe susceptibility of the INVADER-directed cleavage assay to variousmaterials, including but not limited to nucleic acids, glycoproteins andcarbohydrates, likely to be found in a clinical sample, a sample ofhuman saliva was prepared in a manner consistent with practices in theclinical laboratory and the resulting saliva extract was added to theINVADER-directed cleavage assay. The effect of the saliva extract uponthe inhibition of cleavage and upon the specificity of the cleavagereaction was examined.

One and one-half milliliters of human saliva were collected andextracted once with an equal volume of a mixture containingphenol:chloroform:isoamyl alcohol (25:24:1). The resulting mixture wascentrifuged in a microcentrifuge to separate the aqueous and organicphases. The upper, aqueous phase was transferred to a fresh tube.One-tenth volumes of 3 M NaOAc were added and the contents of the tubewere mixed. Two volumes of 100% ethyl alcohol were added to the mixtureand the sample was mixed and incubated at room temperature for 15minutes to allow a precipitate to form. The sample was centrifuged in amicrocentrifuge at 13,000 rpm for 5 minutes and the supernatant wasremoved and discarded. A milky pellet was easily visible. The pellet wasrinsed once with 70% ethanol, dried under vacuum and dissolved in 200 μlof 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA (this constitutes the salivaextract). Each μl of the saliva extract was equivalent to 7.5 μl ofsaliva. Analysis of the saliva extract by scanning ultravioletspectrophotometry showed a peak absorbance at about 260 nm and indicatedthe presence of approximately 45 ng of total nucleic acid per μl ofextract.

The effect of the presence of saliva extract upon the following enzymeswas examined: CLEAVASE BN nuclease, CLEAVASE A/G nuclease and threedifferent lots of DNAPTaq: AmpliTaq® (Perkin Elmer; a recombinant formof DNAPTaq), AmpliTaq® LD (Perkin-Elmer; a recombinant DNAPTaqpreparation containing very low levels of DNA) and Taq DNA polymerase(Fischer). For each enzyme tested, an enzyme/probe mixture was madecomprising the chosen amount of enzyme with 5 pmole of the probeoligonucleotide (SEQ ID NO:32) in 10 μl of 20 mM MOPS (pH 7.5)containing 0.1% each of Tween 20 and NP-40, 4 mM MnCl₂, 100 mM KCl and100 μg/ml BSA. The following amounts of enzyme were used: 25 ng ofCLEAVASE BN prepared as described in Example 8; 2 μl of CLEAVASE A/Gnuclease extract prepared as described in Example 2; 2.25 μl (11.25polymerase units) the following DNA polymerases: AmpliTaq® DNApolymerase (Perkin Elmer); AmpliTaq® D DNA polymerase LD (low DNA; fromPerkin Elmer); Taq DNA polymerase (Fisher Scientific).

For each of the reactions shown in FIG. 33, except for that shown inlane 1, the target DNA (50 fmoles of single-stranded M13mp19 DNA) wascombined with 50 pmole of the INVADER oligonucleotide (SEQ ID NO:35) and5 pmole of the probe oligonucleotide (SEQ ID NO:32); target DNA wasomitted in reaction 1 (lane 1). Reactions 1, 3, 5, 7, 9 and 11 included1.5 μl of saliva extract. These mixtures were brought to a volume of 5μl with distilled water, overlaid with a drop of CHILLOUT evaporationbarrier and brought to 95° C. for 10 minutes. The cleavage reactionswere then started by the addition of 5 μl of the desired enzyme/probemixture; reactions 1, 4 and 5 received CLEAVASE A/G nuclease. Reactions2 and 3 received CLEAVASE BN; reactions 6 and 7 received AmpliTaq®;reactions 8 and 9 received AmpliTaq® LD; and reactions 10 and 11received Taq DNA Polymerase from Fisher Scientific.

The reactions were incubated at 63° C. for 30 minutes and were stoppedby the addition of 6 μl of 95% formamide with 20 mM EDTA and 0.05%marker dyes. Samples were heated to 75° C. for 2 minutes immediatelybefore electrophoresis through a 20% acrylamide gel (19:1 cross-linked),with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.The products of the reactions were visualized by the use of an HitachiFMBIO fluorescence imager, and the results are displayed in FIG. 33.

A pairwise comparison of the lanes shown in FIG. 33 without and with thesaliva extract, treated with each of the enzymes, shows that the salivaextract has different effects on each of the enzymes. While the CLEAVASEBN nuclease and the AmpliTaq® are significantly inhibited from cleavingin these conditions, the CLEAVASE A/G nuclease and AmpliTaq® LD displaylittle difference in the yield of cleaved probe. The preparation of TaqDNA polymerase from Fisher Scientific shows an intermediate response,with a partial reduction in the yield of cleaved product. From thestandpoint of polymerization, the three DNAPTaq variants should beequivalent; these should be the same protein with the same amount ofsynthetic activity. It is possible that the differences observed couldbe due to variations in the amount of nuclease activity present in eachpreparation caused by different handling during purification, or bydifferent purification protocols. In any case, quality control assaysdesigned to assess polymerization activity in commercial DNAPpreparations would be unlikely to reveal variation in the amount ofnuclease activity present. If preparations of DNAPTaq were screened forfull 5′ nuclease activity (i.e., if the 5′ nuclease activity wasspecifically quantitated), it is likely that the preparations woulddisplay sensitivities (to saliva extract) more in line with thatobserved using CLEAVASE A/G nuclease, from which DNAPTaq differs by avery few amino acids.

It is worthy of note that even in the slowed reactions of CLEAVASE BNand the DNAPTaq variants there is no noticeable increase in non-specificcleavage of the probe oligonucleotide due to inappropriate hybridizationor saliva-borne nucleases.

Example 16 Comparison of Additional 5′ Nucleases in the INVADER-DirectedCleavage Assay

A number of eubacterial Type A DNA polymerases (i.e., Pol I type DNApolymerases) have been shown to function as structure specificendonucleases (See, Example 1, and Lyamichev et al., supra). In thisExample, it was demonstrated that the enzymes of this class can also bemade to catalyze the INVADER-directed cleavage of the present invention,albeit not as efficiently as the CLEAVASE enzymes.

CLEAVASE BN nuclease and CLEAVASE A/G nuclease were tested along sidethree different thermostable DNA polymerases: Thermus aquaticus DNApolymerase (Promega), Thermus thermophilus and Thermus flavus DNApolymerases (Epicentre). The enzyme mixtures used in the reactions shownin lanes 1-11 of FIG. 34 contained the following, each in a volume of 5μl: Lane 1: 20 mM MOPS (pH 7.5) with 0.1% each of Tween 20 and NP-40, 4mM MnCl₂, 100 mM KCl; Lane 2: 25 ng of CLEAVASE BN nuclease in the samesolution described for lane 1; Lane 3: 2.25 μl of CLEAVASE A/G nucleaseextract (prepared as described in Example 2), in the same solutiondescribed for lane 1; Lane 4: 2.25 μl of CLEAVASE A/G nuclease extractin 20 mM Tris-Cl, (pH 8.5), 4 mM MgCl₂ and 100 mM KCl; Lane 5: 11.25polymerase units of Taq DNA polymerase in the same buffer described forlane 4; Lane 6: 11.25 polymerase units of Tth DNA polymerase in the samebuffer described for lane 1; Lane 7: 11.25 polymerase units of Tth DNApolymerase in a 2× concentration of the buffer supplied by themanufacturer, supplemented with 4 mM MnCl₂; Lane 8: 11.25 polymeraseunits of Tth DNA polymerase in a 2× concentration of the buffer suppliedby the manufacturer, supplemented with 4 mM MgCl₂; Lane 9: 2.25polymerase units of Tfl DNA polymerase in the same buffer described forlane 1; Lane 10: 2.25 polymerase units of Tfl polymerase in a 2×concentration of the buffer supplied by the manufacturer, supplementedwith 4 mM MnCl₂; Lane 11: 2.25 polymerase units of Tfl DNA polymerase ina 2× concentration of the buffer supplied by the manufacturer,supplemented with 4 mM MgCl₂.

Sufficient target DNA, probe and INVADER for all 11 reactions wascombined into a master mix. This mix contained 550 fmoles ofsingle-stranded M13mp19 target DNA, 550 pmoles of the INVADERoligonucleotide (SEQ ID NO:35) and 55 pmoles of the probeoligonucleotide (SEQ ID NO:32), each as depicted in FIG. 28 c, in 55 μlof distilled water. Five μl of the DNA mixture was dispensed into eachof 11 labeled tubes and overlaid with a drop of CHILLOUT evaporationbarrier. The reactions were brought to 63° C. and cleavage was startedby the addition of 5 μl of the appropriate enzyme mixture. The reactionmixtures were then incubated at 63° C. temperature for 15 minutes. Thereactions were stopped by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. Samples were heated to 90° C for 1 minuteimmediately before electrophoresis through a 20% acrylamide gel (19:1cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate (pH 8.3),1.4 mM EDTA. Following electrophoresis, the products of the reactionswere visualized by the use of an Hitachi FMBIO fluorescence imager, andthe results are displayed in FIG. 34. Examination of the results shownin FIG. 34 demonstrates that all of the 5′ nucleases tested have theability to catalyze INVADER-directed cleavage in at least one of thebuffer systems tested. Although not optimized here, these cleavageagents are suitable for use in the methods of the present invention.

Example 17 The INVADER-Directed Cleavage Assay can Detect Single BaseDifferences in Target Nucleic Acid Sequences

The ability of the INVADER-directed cleavage assay to detect single basemismatch mutations was examined. Two target nucleic acid sequencescontaining CLEAVASE enzyme-resistant phosphorothioate backbones werechemically synthesized and purified by polyacrylamide gelelectrophoresis. Targets comprising phosphorothioate backbones were usedto prevent exonucleolytic nibbling of the target when duplexed with anoligonucleotide. A target oligonucleotide, which provides a targetsequence that is completely complementary to the INVADER oligonucleotide(SEQ ID NO:35) and the probe oligonucleotide (SEQ ID NO:32), containedthe following sequence: 5′-CCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGC-3′(SEQ ID NO:36). A second target sequence containing a single base changerelative to SEQ ID NO:36 was synthesized:5′-CCTTTCGCTCTCTTCCCTTCCTTTCTCGCC ACGTTCGCCGGC-3 (SEQ ID NO:37; thesingle base change relative to SEQ ID NO:36 is shown using bold andunderlined type). The consequent mismatch occurs within the “Z” regionof the target as represented in FIG. 25.

To discriminate between two target sequences that differ by the presenceof a single mismatch), INVADER-directed cleavage reactions wereconducted using two different reaction temperatures (55° C. and 60° C.).Mixtures containing 200 fmoles of either SEQ ID NO:36 or SEQ ID NO:37, 3pmoles of fluorescein-labeled probe oligonucleotide (SEQ ID NO:32), 7.7pmoles of INVADER oligonucleotide (SEQ ID NO:35) and 2 μl of CLEAVASEA/G nuclease extract (prepared as described in Example 2) in 9 μl of 10mM MOPS (pH 7.4) with 50 mM KCl were assembled, covered with a drop ofCHILLOUT evaporation barrier and brought to the appropriate reactiontemperature. The cleavage reactions were initiated by the addition of 1μl of 20 mM MgCl₂. After 30 minutes at either 55° C. or 60° C., 10 μl of95% formamide with 20 mM EDTA and 0.05% marker dyes was added to stopthe reactions. The reaction mixtures where then heated to 90° C. for oneminute prior to loading 4 μl onto 20% denaturing polyacrylamide gels.The resolved reaction products were visualized using a Hitachi FMBIOfluorescence imager. The resulting image is shown in FIG. 35.

In FIG. 35, lanes I and 2 show the products from reactions conducted at55° C.; lanes 3 and 4 show the products from reactions conducted at 60°C. Lanes 1 and 3 contained products from reactions containing SEQ IDNO:36 (perfect match to probe) as the target. Lanes 2 and 4 containedproducts from reactions containing SEQ ID NO:37 (single base mis-matchwith probe) as the target. The target that does not have a perfecthybridization match (i.e., complete complementarity) with the probe willnot bind as strongly (i.e., the T_(m) of that duplex will be lower thanthe T_(m) of the same region if perfectly matched). The resultspresented here show that reaction conditions can be varied to eitheraccommodate the mis-match (e.g., by lowering the temperature of thereaction) or to exclude the binding of the mismatched sequence (e.g., byraising the reaction temperature).

The results shown in FIG. 35 demonstrate that the specific cleavageevent that occurs in INVADER-directed cleavage reactions can beeliminated by the presence of a single base mis-match between the probeoligonucleotide and the target sequence. Thus, reaction conditions canbe chosen so as to exclude the hybridization of mis-matchedINVADER-directed cleavage probes thereby diminishing or even eliminatingthe cleavage of the probe. In an extension of this assay system,multiple cleavage probes, each possessing a separate reporter molecule(i.e., a unique label), could also be used in a single cleavagereaction, to simultaneously probe for two or more variants in the sametarget region. The products of such a reaction would allow not only thedetection of mutations that exist within a target molecule, but wouldalso allow a determination of the relative concentrations of eachsequence (i.e., mutant and wild type or multiple different mutants)present within samples containing a mixture of target sequences. Whenprovided in equal amounts, but in a vast excess (e.g., at least a100-fold molar excess; typically at least 1 pmole of each probeoligonucleotide would be used when the target sequence was present atabout 10 fmoles or less) over the target and used in optimizedconditions. As discussed above, any differences in the relative amountsof the target variants will not affect the kinetics of hybridization, sothe amounts of cleavage of each probe will reflect the relative amountsof each variant present in the reaction.

The results shown in the Example clearly demonstrate that theINVADER-directed cleavage reaction can be used to detect single basedifference between target nucleic acids.

Example 18 The INVADER-Directed Cleavage Reaction is Insensitive toLarge Changes in Reaction Conditions

The results shown above demonstrated that the INVADER-directed cleavagereaction can be used for the detection of target nucleic acid sequencesand that this assay can be used to detect single base difference betweentarget nucleic acids. These results demonstrated that 5′ nucleases(e.g., CLEAVASEBN, CLEAVASE A/G, DNAPTaq, DNAPTth, DNAPTfl) could beused in conjunction with a pair of overlapping oligonucleotides as anefficient way to recognize nucleic acid targets. In the experimentsbelow it is demonstrated that invasive cleavage reaction is relativelyinsensitive to large changes in conditions thereby making the methodsuitable for practice in clinical laboratories.

The effects of varying the conditions of the cleavage reaction wereexamined for their effect(s) on the specificity of the invasive cleavageand the on the amount of signal accumulated in the course of thereaction. To compare variations in the cleavage reaction a “standard”INVADER cleavage reaction was first defined. In each instance, unlessspecifically stated to be otherwise, the indicated parameter of thereaction was varied, while the invariant aspects of a particular testwere those of this standard reaction. The results of these tests areeither shown in FIGS. 38-40, or the results described below.

a) The Standard INVADER-Directed Cleavage Reaction

The standard reaction was defined as comprising 1 fmole of M13mp18single-stranded target DNA (NEB), 5 pmoles of the labeled probeoligonucleotide (SEQ ID NO:38), 10 pmole of the upstream INVADERoligonucleotide (SEQ ID NO:39) and 2 units of CLEAVASE A/G in 10 μl of10 mM MOPS, pH 7.5 with 100 mM KCl, 4 mM MnCl₂, and 0.05% each Tween-20and Nonidet-P40. For each reaction, the buffers, salts and enzyme werecombined in a volume of 5 μl; the DNAs (target and two oligonucleotides)were combined in 5 μl of dH₂O and overlaid with a drop of CHILLOUTevaporation barrier. When multiple reactions were performed with thesame reaction constituents, these formulations were expandedproportionally.

Unless otherwise stated, the sample tubes with the DNA mixtures werewarmed to 61 ° C., and the reactions were started by the addition of 5μl of the enzyme mixture. After 20 minutes at this temperature, thereactions were stopped by the addition of 8 μl of 95%. formamide with 20mM EDTA and 0.05% marker dyes. Samples were heated to 75° C. for 2minutes immediately before electrophoresis through a 20% acrylamide gel(19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA. The products of the reactions were visualized by theuse of an Hitachi FMBIO fluorescence imager. In each case, the uncutprobe material was visible as an intense black band or blob, usually inthe top half of the panel, while the desired products of INVADERspecific cleavage were visible as one or two narrower black bands,usually in the bottom half of the panel. Under some reaction conditions,particularly those with elevated salt concentrations, a secondarycleavage product is also visible (thus generating a doublet). Ladders oflighter grey bands generally indicate either exonuclease nibbling of theprobe oligonucleotide or heat-induced, non-specific breakage of theprobe.

FIG. 37 depicts the annealing of the probe and INVADER oligonucleotidesto regions along the M13mp18 target molecule (the bottom strand). InFIG. 37 only a 52 nucleotide portion of the M13mp18 molecule is shown;this 52 nucleotide sequence is listed in SEQ ID NO:31 (this sequence isidentical in both M13mp18 and M13mp19). The probe oligonucleotide (topstrand) contains a Cy3 amidite label at the 5′ end; the sequence of theprobe is 5′-AGAAAGGAAGGGAAGAAAGCGAAAGGT-3′ (SEQ ID NO:38. The bold typeindicates the presence of a modified base (2′-O—CH₃). Cy3 amidite(Pharmacia) is a indodicarbocyanine dye amidite that can be incorporatedat any position during the synthesis of oligonucleotides; Cy3 fluorescesin the yellow region (excitation and emission maximum of 554 and 568 nm,respectively). The INVADER oligonucleotide (middle strand) has thefollowing sequence: 5′-GCCGGCGAACGTGGCGAGAAAGGA-3′ (SEQ ID NO:39).

b) KCl Titration

FIG. 38 shows the results of varying the KCl concentration incombination with the use of 2 mM MnCl₂, in an otherwise standardreaction. The reactions were performed in duplicate for confirmation ofobservations; the reactions shown in lanes 1 and 2 contained no addedKCl, lanes 3 and 4 contained KCl at 5 mM, lanes 5 and 6 contained 25 mMKCl, lanes 7 and 8 contained 50 mM KCl, lanes 9 and 10 contained 100 mMKCl and lanes 11 and 12 contained 200 mM KCl. These results show thatthe inclusion of KCl allows the generation of a specific cleavageproduct. While the strongest signal is observed at the 100 mM KClconcentration, the specificity of signal in the other reactions with KClat or above 25 mM indicates that concentrations in the full range (i.e.,25-200 mM) may be chosen if it is so desirable for any particularreaction conditions.

As shown in FIG. 38, the INVADER-directed cleavage reaction requires thepresence of salt (e.g., KCl) for effective cleavage to occur. In otherreactions, it has been found that KCl can inhibit the activity ofcertain CLEAVASE enzymes when present at concentrations above about 25mM. For example, in cleavage reactions using the S-60 oligonucleotideshown in FIG. 26, in the absence of primer, the CLEAVASE BN enzyme losesapproximately 50% of its activity in 50 mM KCl. Therefore, the use ofalternative salts in the INVADER-directed cleavage reaction wasexamined. In these experiments, the potassium ion was replaced witheither Na⁺ or Li⁺ or the chloride ion was replaced with glutamic acid.The replacement of KCl with alternative salts is described below inSections c-e.

c) NaCl Titration

NaCl was used in place of KCl at 75, 100, 150 or 200 mM, in combinationwith the use 2 mM MnCl₂, in an otherwise standard reaction. Theseresults showed that NaCl can be used as a replacement for KCl in theINVADER-directed cleavage reaction, with like concentration giving likeresults, (i.e., the presence of NaCl, like KCl, enhances productaccumulation).

d) LiCl Titration

LiCl was used in place of KCl in otherwise standard reactions.Concentrations tested were 25, 50, 75, 100, 150 and 200 mM LiCl. Theresults demonstrated that LiCl can be used as a suitable replacement forKCl in the INVADER-directed cleavage reaction (i.e., the presence ofLiCl, like KCl, enhances product accumulation), in concentrations ofabout 100 mM or higher.

e) KGlu Titration

The results of using a glutamate salt of potassium (KGlu) in place ofthe more commonly used chloride salt (KCl) in reactions performed over arange of temperatures were examined. KGlu has been shown to be a highlyeffective salt source for some enzymatic reactions, showing a broaderrange of concentrations that permit maximum enzymatic activity (Leirmoet al., Biochem., 26:2095 [1987]). The ability of KGlu to facilitate theannealing of the probe and INVADER oligonucleotides to the targetnucleic acid was compared to that of LiCl. In these experiments, thereactions were run for 15 minutes, rather than the standard 20 minutes,in standard reactions that replaced KCl 200 mM, 300 mM or 400 mM KGlu.The reactions were run at 65° C., 67° C., 69° C. or 71° C. The resultsshowed demonstrated that KGlu was very effective as a salt in theinvasive cleavage reactions, with full activity apparent even at 400 mMKGlu, though at the lowest temperature cleavage was reduced by about 30%at 300 mM KGlu, and by about 90% to 400 mM KGlu.

f) MnCl₂ and MgCl₂ Titration and Ability to Replace MnCl₂ with MgCl₂

In some instances it may be desirable to perform the invasive cleavagereaction in the presence of Mg²⁺, either in addition to, or in place ofMn²⁺ as the necessary divalent cation required for activity of theenzyme employed. For example, some common methods of preparing DNA frombacterial cultures or tissues use MgCl₂ in solutions that are used tofacilitate the collection of DNA by precipitation. In addition, elevatedconcentrations (i.e., greater than 5 mM) of divalent cation can be usedto facilitate hybridization of nucleic acids, in the same way that themonovalent salts were used above, thereby enhancing the invasivecleavage reaction. In this experiment, the tolerance of the invasivecleavage reaction was examined for 1) the substitution of MgCl₂ forMnCl₂ and for the ability to produce specific product in the presence ofincreasing concentrations of MgCl₂ and MnCl₂.

FIG. 39 shows the results of either varying the concentration of MnCl₂from 2 mM to 8 mM, replacing the MnCl₂ with MgCl₂ at 2 to 4 mM, or ofusing these components in combination in an otherwise standard reaction.The reactions analyzed in lanes 1 and 2 contained 2 mM each MnCl₂ andMgCl₂, lanes 3 and 4 contained 2 mM MnCl₂ only, lanes 5 and 6 contained3 mM MnCl₂, lanes 7 and 8 contained 4 mM MnCl₂, lanes 9 and 10 contained8 mM MnCl₂. The reactions analyzed in lanes 11 and 12 contained 2 mMMgCl₂ and lanes 13 and 14 contained 4 mM MgCl₂. These results show thatboth MnCl₂ and MgCl₂ can be used as the necessary divalent cation toenable the cleavage activity of the CLEAVASE A/G enzyme in thesereactions and that the invasive cleavage reaction can tolerate a broadrange of concentrations of these components.

In addition to examining the effects of the salt environment on the rateof product accumulation in the invasive cleavage reaction, the use ofreaction constituents shown to be effective in enhancing nucleic acidhybridization in either standard hybridization assays (e.g., blothybridization) or in ligation reactions was examined. These componentsmay act as volume excluders, increasing the effective concentration ofthe nucleic acids of interest and thereby enhancing hybridization, orthey may act as charge-shielding agents to minimize repulsion betweenthe highly charged backbones of the nucleic acids strands. The resultsof these experiments are described in Sections g and h below.

g) Effect of CTAB Addition

The polycationic detergent cetyltrietheylammonium bromide (CTAB) hasbeen shown to dramatically enhance hybridization of nucleic acids(Pontius and Berg, Proc. Natl. Acad. Sci. USA 88:8237 [1991]). Theeffect of adding the detergent CTAB in concentrations from 100 mM to ImM to invasive cleavage reactions in which 150 mM LiCl was used in placeof the KCl in otherwise standard reactions was also investigated. Theseresults showed that 200 mM CTAB may have a very moderate enhancingeffect under these reaction conditions, and the presence of CTAB inexcess of about 500 μM was inhibitory to the accumulation of specificcleavage product.

h) Effect of PEG Addition

The effect of adding polyethylene glycol (PEG) at 4.8 or 12% (w/v)concentrations to otherwise standard reactions was also examined. Theeffects of increasing the reaction temperature of the PEG-containingreactions was examined by performing duplicate sets of PEG titrationreactions at 61° C. and 65° C. The results showed that at allpercentages tested, and at both temperatures tested, the inclusion ofPEG substantially eliminated the production of specific cleavageproduct.

In addition to, the presence of 1× Denhardts in the reaction mixture wasfound to have no adverse effect upon the cleavage reaction (50×Denhardts contains per 500 ml: 5 g Ficoll, 5 g polyvinylpyrrolidone, 5 gBSA). Further, the presence of each component of Denhardt's was examinedindividually (i.e., Ficoll alone, polyvinylpyrrolidone alone, BSA alone)for the effect upon the INVADER-directed cleavage reaction; no adverseeffect was observed.

i) Effect of the Addition of Stabilizing Agents

Another approach to enhancing the output of the invasive cleavagereaction is to enhance the activity of the enzyme employed, either byincreasing its stability in the reaction environment or by increasingits turnover rate. Without regard to the precise mechanism by whichvarious agents operate in the invasive cleavage reaction, a number ofagents commonly used to stabilize enzymes during prolonged storage weretested for the ability to enhance the accumulation of specific cleavageproduct in the invasive cleavage reaction.

The effects of adding glycerol at 15% and of adding the detergentsTween-20 and Nonidet-P40 at 1.5%, alone or in combination, in otherwisestandard reactions were also examined. The results demonstrated thatunder these conditions these adducts had little or no effect on theaccumulation of specific cleavage product.

The effects of adding gelatin to reactions in which the salt identityand concentration were varied from the standard reaction were alsoinvestigated. The results demonstrated that in the absence of salt thegelatin had a moderately enhancing effect on the accumulation ofspecific cleavage product, but when either salt (KCl or LiCl) was addedto reactions performed under these conditions, increasing amounts ofgelatin reduced the product accumulation.

j) Effect of Adding Large Amounts of Non-Target Nucleic Acid

In detecting specific nucleic acid sequences within samples, it isimportant to determine if the presence of additional genetic material(i.e., non-target nucleic acids) will have a negative effect on thespecificity of the assay. In this experiment, the effect of includinglarge amounts of non-target nucleic acid, either DNA or RNA, on thespecificity of the invasive cleavage reaction was examined. The data wasexamined for either an alteration in the expected site of cleavage, orfor an increase in the nonspecific degradation of the probeoligonucleotide.

FIG. 40 shows the effects of adding non-target nucleic acid (e.g.,genomic DNA or tRNA) to an invasive cleavage reaction performed at 65°C., with 150 mM LiCl in place of the KCl in the standard reaction. Thereactions assayed in lanes 1 and 2 contained 235 and 470 ng of genomicDNA, respectively. The reactions analyzed in lanes 3, 4, 5 and 6contained 100 ng, 200 ng, 500 ng and 1 μg of tRNA, respectively. Lane 7represents a control reaction that contained no added nucleic acidbeyond the amounts used in the standard reaction. The results shown inFIG. 40 demonstrate that the inclusion of non-target nucleic acid inlarge amounts could visibly slow the accumulation of specific cleavageproduct (while not limiting the invention to any particular mechanism,it is thought that the additional nucleic acid competes for binding ofthe enzyme with the specific reaction components). In additionalexperiments it was found that the effect of adding large amounts ofnon-target nucleic acid can be compensated for by increasing the enzymein the reaction. The data shown in FIG. 40 also demonstrate that a keyfeature of the invasive cleavage reaction, the specificity of thedetection, was not compromised by the presence of large amounts ofnon-target nucleic acid.

In addition to the data presented above, invasive cleavage reactionswere run with succinate buffer at pH 5.9 in place of the MOPS bufferused in the “standard” reaction; no adverse effects were observed.

The data shown in FIGS. 38-40 and described above demonstrate that theinvasive cleavage reaction can be performed using a wide variety ofreaction conditions and is therefore suitable for practice in clinicallaboratories.

Example 19 Detection of RNA Targets by INVADER-Directed Cleavage

In addition to the clinical need to detect specific DNA sequences forinfectious and genetic diseases, there is a need for technologies thatcan quantitatively detect target nucleic acids that are composed of RNA.For example, a number of viral agents, such as hepatitis C virus (HCV)and human immunodeficiency virus (HIV) have RNA genomic material, thequantitative detection of which can be used as a measure of viral loadin a patient sample. Such information can be of critical diagnostic orprognostic value.

Hepatitis C virus (HCV) infection is the predominant cause ofpost-transfusion non-A, non-B (NANB) hepatitis around the world. Inaddition, HCV is the major etiologic agent of hepatocellular carcinoma(HCC) and chronic liver disease world wide. The genome of HCV is a small(9.4 kb) RNA molecule. In studies of transmission of HCV by bloodtransfusion it has been found the presence of HCV antibody, as measuredin standard immunological tests, does not always correlate with theinfectivity of the sample, while the presence of HCV RNA in a bloodsample strongly correlates with infectivity. Conversely, serologicaltests may remain negative in immunosuppressed infected individuals,while HCV RNA may be easily detected (Cuthbert, Clin. Microbiol. Rev.,7:505 [1994]).

The need for and the value of developing a probe-based assay for thedetection the HCV RNA is clear. The polymerase chain reaction has beenused to detect HCV in clinical samples, but the problems associated withcarry-over contamination of samples has been a concern. Direct detectionof the viral RNA without the need to perform either reversetranscription or amplification would allow the elimination of several ofthe points at which existing assays may fail.

The genome of the positive-stranded RNA hepatitis C virus comprisesseveral regions including 5′ and 3′ noncoding regions (i.e., 5′ and 3′untranslated regions) and a polyprotein coding region that encodes thecore protein (C), two envelope glycoproteins (E1 and E2/NS1) and sixnonstructural glycoproteins (NS2-NS5b). Molecular biological analysis ofthe HCV genome has showed that some regions of the genome are veryhighly conserved between isolates, while other regions are fairlyrapidly changeable. The 5′ noncoding region (NCR) is the most highlyconserved region in the HCV. These analyses have allowed these virusesto be divided into six basic genotype groups, and then furtherclassified into over a dozen sub-types (the nomenclature and division ofHCV genotypes is evolving; see Altamirano et al., J. Infect. Dis.,171:1034 (1995) for a recent classification scheme).

In order to develop a rapid and accurate method of detecting HCV presentin infected individuals, the ability of the INVADER-directed cleavagereaction to detect HCV RNA was examined. Plasmids containing DNA derivedfrom the conserved 5′-untranslated region of six different HCV RNAisolates were used to generate templates for in vitro transcription. TheHCV sequences contained within these six plasmids represent genotypes 1(four sub-types represented; 1a, 1b, 1c, and Δ1c), 2, and 3. Thenomenclature of the HCV genotypes used herein is that of Simmonds et al.(as described in Altamirano et al., supra). The Δ1c subtype was used inthe model detection reaction described below.

a) Generation of Plasmids Containing HCV Sequences

Six DNA fragments derived from HCV were generated by RT-PCR using RNAextracted from serum samples of blood donors; these PCR fragments were agift of Dr. M. Altamirano (University of British Columbia, Vancouver).These PCR fragments represent HCV sequences derived from HCV genotypes1a, 1b, 1c, Δ1c, 2c and 3a.

The RNA extraction, reverse transcription and PCR were performed usingstandard techniques (Altamirano et al., supra). Briefly, RNA wasextracted from 100 μl of serum using guanidine isothiocyanate, sodiumlauryl sarkosate and phenol-chloroform (Inchauspe et al., Hepatol.,14:595 [1991]). Reverse transcription was performed according to themanufacturer's instructions using a GeneAmp rTh reverse transcriptaseRNA PCR kit (Perkin-Elmer) in the presence of an external antisenseprimer, HCV342. The sequence of the HCV342 primer is5′-GGTTTTTCTTTGAGGTTTAG-3′ (SEQ ID NO:40). Following termination of theRT reaction, the sense primer HCV7 (5′-GCGACACTCCACCATAGAT-3′ [SEQ IDNO:41]) and magnesium were added and a first PCR was performed. Aliquotsof the first PCR products were used in a second (nested) PCR in thepresence of primers HCV46 (5′-CTGTCTTCACGCAGAAAGC-3′ [SEQ ID NO:42]) andHCV308 [5′-GCACGGT CTACGAGACCTC-3′ [SEQ ID NO:43]). The PCRs produced a281 bp product that corresponds to a conserved 5′ noncoding region (NCR)region of HCV between positions -284 and -4 of the HCV genome(Altramirano et al., supra).

The six 281 bp PCR fragments were used directly for cloning or they weresubjected to an additional amplification step using a 50 μl PCRcomprising approximately 100 fmoles of DNA, the HCV46 and HCV308 primersat 0.1 μM, 100 μM of all four dNTPs and 2.5 units of Taq DNA polymerasein a buffer containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂and 0.1% Tween 20. The PCRs were cycled 25 times at 96° C. for 45 sec.,55° C. for 45 sec. and 72° C. for 1 min. Two microliters of either theoriginal DNA samples or the reamplified PCR products were used forcloning in the linear pT7Blue T-vector (Novagen) according tomanufacturer's protocol. After the PCR products were ligated to thepT7Blue T-vector, the ligation reaction mixture was used to transformcompetent JM109 cells (Promega). Clones containing the pT7Blue T-vectorwith an insert were selected by the presence of colonies having a whitecolor on LB plates containing 40 μg/ml X-Gal, 40 μg/ml IPTG and 50 μg/mlampicillin. Four colonies for each PCR sample were picked and grownovernight in 2 ml LB media containing 50 μg/ml carbenicillin. PlasmidDNA was isolated using the following alkaline miniprep protocol. Cellsfrom 1.5 ml of the overnight culture were collected by centrifugationfor 2 min. in a microcentrifuge (14K rpm), the supernatant was discardedand the cell pellet was resuspended in 50 μl TE buffer with 10 μg/mlRNAse A (Pharmacia). One hundred microliters of a solution containing0.2 N NaOH, 1% SDS was added and the cells were lysed for 2 min. Thelysate was gently mixed with 100 μl of 1.32 M potassium acetate, pH 4.8,and the mixture was centrifuged for 4 min. in a microcentrifuge (14Krpm); the pellet comprising cell debris was discarded. Plasmid DNA wasprecipitated from the supernatant with 200 μl ethanol and pelleted bycentrifugation a microcentrifuge (14K rpm). The DNA pellet was air driedfor 15 min. and was then redissolved in 50 μl TE buffer (10 mM Tris-HCl,pH 7.8, 1 mM EDTA).

b) Reamplification of HCV Clones to Add the Phage T7 Promoter forSubsequent In Vitro Transcription

To ensure that the RNA product of transcription had a discrete 3′ end itwas necessary to create linear transcription templates that stopped atthe end of the HCV sequence. These fragments were conveniently producedusing the PCR to reamplify the segment of the plasmid containing thephage promoter sequence and the HCV insert. For these studies, the cloneof HCV type Δ1c was reamplified using a primer that hybridizes to the T7promoter sequence: 5′-TAATACGACTCACTATAGGG-3′ (SEQ ID NO:44; “the T7promoter primer”) (Novagen) in combination with the 3′ terminalHCV-specific primer HCV308 (SEQ ID NO:43). For these reactions, 1 μl ofplasmid DNA (approximately 10 to 100 ng) was reamplified in a 200 μl PCRusing the T7 and HCV308 primers as described above with the exceptionthat 30 cycles of amplification were employed. The resulting ampliconwas 354 bp in length. After amplification the PCR mixture wastransferred to a fresh 1.5 ml microcentrifuge tube, the mixture wasbrought to a final concentration of 2 M NH₄OAc, and the products wereprecipitated by the addition of one volume of 100% isopropanol.Following a 10 min. incubation at room temperature, the precipitateswere collected by centrifugation, washed once with 80% ethanol and driedunder vacuum. The collected material was dissolved in 100 μlnuclease-free distilled water (Promega).

Segments of RNA were produced from this amplicon by in vitrotranscription using the RiboMAX™ Large Scale RNA Production System(Promega) in accordance with the manufacturer's instructions, using 5.3∥g of the amplicon described above in a 100 μl reaction. Thetranscription reaction was incubated for 3.75 hours, after which the DNAtemplate was destroyed by the addition of 5-6 μl of RQ1 RNAse-free DNAse(1 unit/μl) according to the RiboMAX™ kit instructions. The reaction wasextracted twice with phenol/chloroform/isoamyl alcohol (50:48:2) and theaqueous phase was transferred to a fresh microcentrifuge tube. The RNAwas then collected by the addition of 10 μl of 3M NH₄OAc, pH 5.2 and 110μl of 100% isopropanol. Following a 5 min. incubation at 4° C., theprecipitate was collected by centrifugation, washed once with 80%ethanol and dried under vacuum. The sequence of the resulting RNAtranscript (HCV 1.1 transcript) is listed in SEQ ID NO:45.

c) Detection of the HCV1.1 Transcript in the INVADER-Directed CleavageAssay

Detection of the HCV1.1 transcript was tested in the INVADER-directedcleavage assay using an HCV-specific probe oligonucleotide(5′-CCGGTCGTCCTGGCAAT XCC-3′ [SEQ ID NO:46]); X indicates the presenceof a fluorescein dye on an abasic linker) and an HCV-specific INVADERoligonucleotide (5′-GTTTATCCAAGAAAGGAC CCGGTC-3′ [SEQ ID NO:47]) thatcauses a 6-nucleotide invasive cleavage of the probe.

Each 10 μl of reaction mixture comprised 5 pmole of the probeoligonucleotide (SEQ ID NO:46) and 10 pmole of the INVADERoligonucleotide (SEQ ID NO:47) in a buffer of 10 mM MOPS, pH 7.5 with 50mM KCl, 4 mM MnCl₂, 0.05% each Tween-20 and Nonidet-P40 and 7.8 unitsRNasin® ribonuclease inhibitor (Promega). The cleavage agents employedwere CLEAVASE A/G (used at 5.3 ng/10 μl reaction) or DNAPTth (used at 5polymerase units/10 μl reaction). The amount of RNA target was varied asindicated below. When RNAse treatment is indicated, the target RNAs werepre-treated with 10 μg of RNase A (Sigma) at 37° C. for 30 min. todemonstrate that the detection was specific for the RNA in the reactionand not due to the presence of any residual DNA template from thetranscription reaction. RNase-treated aliquots of the HCV RNA were useddirectly without intervening purification.

For each reaction, the target RNAs were suspended in the reactionsolutions as described above, but lacking the cleavage agent and theMnCl₂ for a final volume of 10 μl, with the INVADER and probe at theconcentrations listed above. The reactions were warmed to 46° C. and thereactions were started by the addition of a mixture of the appropriateenzyme with MnCl₂. After incubation for 30 min. at 46° C., the reactionswere stopped by the addition of 8 μl of 95% formamide, 10 mM EDTA and0.02% methyl violet (methyl violet loading buffer). Samples were thenresolved by electrophoresis through a 15% denaturing polyacrylamide gel(19:1 cross-linked), containing 7 M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA. Following electrophoresis, the labeledreaction products were visualized using the FMBIO-100 Image Analyzer(Hitachi), with the resulting imager scan shown in FIG. 41.

In FIG. 41, the samples analyzed in lanes 1-4 contained 1 pmole of theRNA target, the reactions shown in lanes 5-8 contained 100 fmoles of theRNA target and the reactions shown in lanes 9-12 contained 10 fmoles ofthe RNA target. All odd-numbered lanes depict reactions performed usingCLEAVASE A/G enzyme and all even-numbered lanes depict reactionsperformed using DNAPTth. The reactions analyzed in lanes 1, 2, 5, 6, 9and 10 contained RNA that had been pre-digested with RNase A. These datademonstrate that the invasive cleavage reaction efficiently detects RNAtargets and further, the absence of any specific cleavage signal in theRNase-treated samples confirms that the specific cleavage product seenin the other lanes is dependent upon the presence of input RNA.

Example 20 The Fate of the Target RNA in the INVADER-Directed CleavageReaction

In this Example, the fate of the RNA target in the INVADER-directedcleavage reaction was examined. As shown above in Example 1D, when RNAsare hybridized to DNA oligonucleotides, the 5′ nucleases associated withDNA polymerases can be used to cleave the RNAs; such cleavage can besuppressed when the 5′ arm is long or when it is highly structured(Lyamichev et al, Science 260:778 [1993], and U.S. Pat. No. 5,422,253,the disclosure of which is herein incorporated by reference). In thisexperiment, the extent to which the RNA target would be cleaved by thecleavage agents when hybridized to the detection oligonucleotides (i.e.,the probe and INVADER oligonucleotides) was examined using reactionssimilar to those described in Example 20, performed usingfluorescein-labeled RNA as a target.

Transcription reactions were performed as described in Example 19 withthe exception that 2% of the UTP in the reaction was replaced withfluorescein-12-UTP (Boehringer Mannheim) and 5.3 μg of the amplicon wasused in a 100 μl reaction. The transcription reaction was incubated for2.5 hours, after which the DNA template was destroyed by the addition of5-6 μl of RQ1 RNAse-free DNAse (1 unit/μl) according to the RiboMAX™ kitinstructions. The organic extraction was omitted and the RNA wascollected by the addition of 10 μl of 3M NaOAc, pH 5.2 and 110 μl of100% isopropanol. Following a 5 min. incubation at 4° C., theprecipitate was collected by centrifugation, washed once with 80%ethanol and dried under vacuum. The resulting RNA was dissolved in 100μl of nuclease-free water. Half (i.e., 50%) of the sample was purifiedby electrophoresis through a 8% denaturing polyacrylamide gel (19:1cross-linked), containing 7 M urea, in a buffer of 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA. The gel slice containing the full-length material wasexcised and the RNA was eluted by soaking the slice overnight at 4° C.in 200 μl of 10 mM Tris-Cl, pH 8.0, 0.1 mM EDTA and 0.3 M NaOAc. The RNAwas then precipitated by the addition of 2.5 volumes of 100% ethanol.After incubation at −20° C. for 30 min., the precipitates were recoveredby centrifugation, washed once with 80% ethanol and dried under vacuum.The RNA was dissolved in 25 μl of nuclease-free water and thenquantitated by UV absorbance at 260 nm.

Samples of the purified RNA target were incubated for 5 or 30 min. inreactions that duplicated the CLEAVASE A/G and DNAPTth INVADER reactionsdescribed in Example 20 with the exception that the reactions lackedprobe and INVADER oligonucleotides. Subsequent analysis of the productsshowed that the RNA was very stable, with a very slight background ofnon-specific degradation, appearing as a gray background in the gellane. The background was not dependent on the presence of enzyme in thereaction.

INVADER detection reactions using the purified RNA target were performedusing the probe/INVADER pair described in Example 19 (SEQ ID NOS:46 and47). Each reaction included 500 fmole of the target RNA, 5 pmoles of thefluorescein-labeled probe and 10 pmoles of the INVADER oligonucleotidein a buffer of 10 mM MOPS, pH 7.5 with 150 mM LiCl, 4 mM MnCl₂, 0.05%each Tween-20 and Nonidet-P40 and 39 units RNAsin® (Promega). Thesecomponents were combined and warmed to 50° C. and the reactions werestarted by the addition of either 53 ng of CLEAVASE A/G or 5 polymeraseunits of DNAPTth. The final reaction volume was 10 μl. After 5 min at50° C., 5 μl aliquots of each reaction were removed to tubes containing4 μl of 95% formamide, 10 mM EDTA and 0.02% methyl violet. The remainingaliquot received a drop of CHILLOUT evaporation barrier and wasincubated for an additional 25 min. These reactions were then stopped bythe addition of 4 μl of the above formamide solution. The products ofthese reactions were resolved by electrophoresis through separate 20%denaturing polyacrylamide gels (19:1 cross-linked), containing 7 M urea,in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Followingelectrophoresis, the labeled reaction products were visualized using theFMBIO-100 Image Analyzer (Hitachi), with the resulting imager scansshown in FIGS. 42A (5 min reactions) and 42B (30 min. reactions).

In FIG. 53 the target RNA is seen very near the top of each lane, whilethe labeled probe and its cleavage products are seen just below themiddle of each panel. The FMBIO-100 Image Analyzer was used toquantitate the fluorescence signal in the probe bands. In each panel,lane 1 contains products from reactions performed in the absence of acleavage agent, lane 2 contains products from reactions performed usingCLEAVASE A/G and lane 3 contains products from reactions performed usingDNAPTth.

Quantitation of the fluorescence signal in the probe bands revealed thatafter a 5 min. incubation, 12% or 300 fmole of the probe was cleaved bythe CLEAVASE A/G and 29% or 700 fmole was cleaved by the DNAPTth. Aftera 30 min. incubation, CLEAVASE A/G had cleaved 32% of the probemolecules and DNAPTth had cleaved 70% of the probe molecules. (Theimages shown in FIGS. 42A and 42B were printed with the intensityadjusted to show the small amount of background from the RNAdegradation, so the bands containing strong signals are saturated andtherefore these images do not accurately reflect the differences inmeasured fluorescence) The data shown in FIG. 42 clearly shows that,under invasive cleavage conditions, RNA molecules are sufficientlystable to be detected as a target and that each RNA molecule can supportmany rounds of probe cleavage.

Example 21 Titration of Target RNA in the INVADER-Directed CleavageAssay

One of the primary benefits of the INVADER-directed cleavage assay as ameans for detection of the presence of specific target nucleic acids isthe correlation between the amount of cleavage product generated in aset amount of time and the quantity of the nucleic acid of interestpresent in the reaction. The benefits of quantitative detection of RNAsequences was discussed in Example 19. In this Example, the quantitativenature of the detection assay was demonstrated through the use ofvarious amounts of target starting material. In addition todemonstrating the correlation between the amounts of input target andoutput cleavage product, these data graphically show the degree to whichthe RNA target can be recycled in this assay The RNA target used inthese reactions was the fluorescein-labeled material described inExample 20 (i.e., SEQ ID NO:45). Because the efficiency of incorporationof the fluorescein-12-UTP by the T7 RNA polymerase was not known, theconcentration of the RNA was determined by measurement of absorbance at260 nm, not by fluorescence intensity. Each reaction comprised 5 pmolesof the fluorescein-labeled probe (SEQ ID NO:46) and 10 pmoles of theINVADER oligonucleotide (SEQ ID NO:47) in a buffer of 10 mM MOPS, pH 7.5with 150 mM LiCl, 4 mM MnCl₂, 0.05% each Tween-20 and Nonidet-P40 and 39units of RNAsin® (Promega). The amount of target RNA was varied from 1to 100 fmoles, as indicated below. These components were combined,overlaid with CHILLOUT evaporation barrier and warmed to 50° C.; thereactions were started by the addition of either 53 ng of CLEAVASE A/Gor 5 polymerase units of DNAPTth, to a final reaction volume of 10 μl.After 30 minutes at 50° C., reactions were stopped by the addition of 8μl of 95% formamide, 10 mM EDTA and 0.02% methyl violet. The unreactedmarkers in lanes 1 and 2 were diluted in the same total volume (18 μl).The samples were heated to 90° C. for 1 minute and 2.5 μl of each ofthese reactions were resolved by electrophoresis through a 20%denaturing polyacrylamide gel (19:1 cross link) with 7M urea in a bufferof 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA, and the labeled reactionproducts were visualized using the FMBIO-100 Image Analyzer (Hitachi),with the resulting imager scans shown in FIG. 43.

In FIG. 43, lanes 1 and 2 show 5 pmoles of uncut probe and 500 fmoles ofuntreated RNA, respectively. The probe is the very dark signal near themiddle of the panel, while the RNA is the thin line near the top of thepanel. These RNAs were transcribed with a 2% substitution offluorescein-12-UTP for natural UTP in the transcription reaction. Theresulting transcript contains 74 U residues, which would give an averageof 1.5 fluorescein labels per molecule. With one tenth the molar amountof RNA loaded in lane 2, the signal in lane 2 should be approximatelyone seventh (0.15×) the fluorescence intensity of the probe in lane 1.Measurements indicated that the intensity was closer to one fortieth,indicating an efficiency of label incorporation of approximately 17%.Because the RNA concentration was verified by A260 measurement this doesnot alter the experimental observations below, but it should be notedthat the signal from the RNA and the probes does not accurately reflectthe relative amounts in the reactions.

The reactions analyzed in lanes 3 through 7 contained 1, 5, 10, 50 and100 fmoles of target, respectively, with cleavage of the probeaccomplished by CLEAVASE A/G. The reactions analyzed in lanes 8 through12 repeated the same array of target amounts, with cleavage of the probeaccomplished by DNAPTth. The boxes seen surrounding the product bandsshow the area of the scan in which the fluorescence was measured foreach reaction. The number of fluorescence units detected within each boxis indicated below each box; background florescence was also measured.

It can be seen by comparing the detected fluorescence in each lane thatthe amount of product formed in these 30 minute reactions can becorrelated to the amount of target material. The accumulation of productunder these conditions is slightly enhanced when DNAPTth is used as thecleavage agent, but the correlation with the amount of target presentremains. This demonstrates that the INVADER assay can be used as a meansof measuring the amount of target RNA within a sample.

Comparison of the fluorescence intensity of the input RNA with that ofthe cleaved product shows that the INVADER-directed cleavage assaycreates signal in excess of the amount of target, so that the signalvisible as cleaved probe is far more intense than that representing thetarget RNA. This further confirms the results described in Example 20,in which it was demonstrated that each RNA molecule could be used manytimes.

Example 22 Detection of DNA by Charge Reversal

The detection of specific targets is achieved in the INVADER-directedcleavage assay by the cleavage of the probe oligonucleotide. In additionto the methods described in the preceding Examples, the cleaved probemay be separated from the uncleaved probe using the charge reversaltechnique described below. This novel separation technique is related tothe observation that positively charged adducts can affect theelectrophoretic behavior of small oligonucleotides because the charge ofthe adduct is significant relative to charge of the whole complex.Observations of aberrant mobility due to charged adducts have beenreported in the literature, but in all cases found, the applicationspursued by other scientists have involved making oligonucleotides largerby enzymatic extension. As the negatively charged nucleotides are addedon, the positive influence of the adduct is reduced to insignificance.As a result, the effects of positively charged adducts have beendismissed and have received infinitesimal notice in the existingliterature.

This observed effect is of particular utility in assays based on thecleavage of DNA molecules. When an oligonucleotide is shortened throughthe action of a CLEAVASE enzyme or other cleavage agent, the positivecharge can be made to not only significantly reduce the net negativecharge, but to actually override it, effectively “flipping” the netcharge of the labeled entity. This reversal of charge allows theproducts of target-specific cleavage to be partitioned from uncleavedprobe by extremely simple means. For example, the products of cleavagecan be made to migrate towards a negative electrode placed at any pointin a reaction vessel, for focused detection without gel-basedelectrophoresis. When a slab gel is used, sample wells can be positionedin the center of the gel, so that the cleaved and uncleaved probes canbe observed to migrate in opposite directions. Alternatively, atraditional vertical gel can be used, but with the electrodes reversedrelative to usual DNA gels (i.e., the positive electrode at the top andthe negative electrode at the bottom) so that the cleaved moleculesenter the gel, while the uncleaved disperse into the upper reservoir ofelectrophoresis buffer.

An additional benefit of this type of readout is that the absolutenature of the partition of products from substrates means that anabundance of uncleaved probe can be supplied to drive the hybridizationstep of the probe-based assay, yet the unconsumed probe can besubtracted from the result to reduce background.

Through the use of multiple positively charged adducts, syntheticmolecules can be constructed with sufficient modification that thenormally negatively charged strand is made nearly neutral. When soconstructed, the presence or absence of a single phosphate group canmean the difference between a net negative or a net positive charge.This observation has particular utility when one objective is todiscriminate between enzymatically generated fragments of DNA, whichlack a 3′ phosphate, and the products of thermal degradation, whichretain a 3′ phosphate (and thus two additional negative charges).

a) Characterization of the Products of Thermal Breakage of DNAOligonucleotides

Thermal degradation of DNA probes results in high background that canobscure signals generated by specific enzymatic cleavage, decreasing thesignal-to-noise ratio. To better understand the nature of DNA thermaldegradation products, the 5′ tetrachloro-fluorescein (TET)-labeledoligonucleotides 78 (SEQ ID NO:48) and 79 (SEQ ID NO:49) (100 pmoleeach) were incubated in 50 μl 10 mM NaCO₃ (pH 10.6), 50 mM NaCl at 90°C. for 4 hours. To prevent evaporation of the samples, the reactionmixture was overlaid with 50 μl of CHILLOUT liquid wax. The reactionswere then divided in two equal aliquots (A and B). Aliquot A was mixedwith 25 μl of methyl violet loading buffer and Aliquot B wasdephosphorylated by addition of 2.5 μl of 100 mM MgCl₂ and 1 μl of 1unit/μl Calf Intestinal Alkaline Phosphatase (CIAP) (Promega), withincubation at 37° C. for 30 min. after which 25 μl of methyl violetloading buffer was added. One microliter of each sample was resolved byelectrophoresis through a 12% polyacrylamide denaturing gel and imagedas described in Example 21; a 585 nm filter was used with the FMBIOImage Analyzer. The resulting imager scan is shown in FIG. 44.

In FIG. 44, lanes 1-3 contain the TET-labeled oligonucleotide 78 andlanes 4-6 contain the TET-labeled oligonucleotides 79. Lanes 1 and 4contain products of reactions that were not heat treated. Lanes 2 and 5contain products from reactions that were heat treated and lanes 3 and 6contain products from reactions that were heat treated and subjected tophosphatase treatment.

As shown in FIG. 44, heat treatment causes significant breakdown of the5′-TET-labeled DNA, generating a ladder of degradation products (FIG.44, lanes 2, 3, 5 and 6). Band intensities correlate with purine andpyrimidine base positioning in the oligonucleotide sequences, indicatingthat backbone hydrolysis may occur through formation of abasicintermediate products that have faster rates for purines than forpyrimidines (Lindahl and Karlström, Biochem., 12:5151 [1973]).

Dephosphorylation decreases the mobility of all products generated bythe thermal degradation process, with the most pronounced effectobserved for the shorter products (FIG. 44, lanes 3 and 6). Thisdemonstrates that thermally degraded products possess a 3′ end terminalphosphoryl group that can be removed by dephosphorylation with CIAP.Removal of the phosphoryl group decreases the overall negative charge by2. Therefore, shorter products that have a small number of negativecharges are influenced to a greater degree upon the removal of twocharges. This leads to a larger mobility shift in the shorter productsthan that observed for the larger species.

The fact that the majority of thermally degraded DNA products contain 3′end phosphate groups and CLEAVASE enzyme-generated products do notallowed the development of simple isolation methods for productsgenerated in the INVADER-directed cleavage assay. The extra two chargesfound in thermal breakdown products do not exist in the specificcleavage products. Therefore, if one designs assays that producespecific products that contain a net positive charge of one or two, thensimilar thermal breakdown products will either be negative or neutral.The difference can be used to isolate specific products by reversecharge methods as shown below.

b) Dephosphorylation of Short Amino-Modified Oligonucleotides canReverse the Net Charge of the Labeled Product

To demonstrate how oligonucleotides can be transformed from net negativeto net positively charged compounds, the four short amino-modifiedoligonucleotides labeled 70, 74, 75 and 76 and shown in FIGS. 45-47 weresynthesized (FIG. 45 shows both oligonucleotides 70 and 74). All fourmodified oligonucleotides possess Cy-3 dyes positioned at the 5′-end,which individually are positively charged under reaction and isolationconditions described in this Example. Compounds 70 and 74 contain twoamino modified thymidines that, under reaction conditions, displaypositively charged R-NH₃ ⁺ groups attached at the C5 position through aC₁₀ or C₆ linker, respectively. Because compounds 70 and 74 are 3′-endphosphorylated, they consist of four negative charges and three positivecharges. Compound 75 differs from 74 in that the internal C₆ aminomodified thymidine phosphate in 74 is replaced by a thymidine methylphosphonate. The phosphonate backbone is uncharged and so there are atotal of three negative charges on compound 75. This gives compound 75 anet negative one charge. Compound 76 differs from 70 in that theinternal amino modified thymidine is replaced by an internal cytosinephosphonate. The pK_(a) of the N3 nitrogen of cytosine can be from 4 to7. Thus, the net charges of this compound, can be from −1 to 0 dependingon the pH of the solution. For the simplicity of analysis, each group isassigned a whole number of charges, although it is realized that,depending on the pK_(a) of each chemical group and ambient pH, a realcharge may differ from the whole number assigned. It is assumed thatthis difference is not significant over the range of pHs used in theenzymatic reactions studied here.

Dephosphorylation of these compounds, or the removal of the 3′ endterminal phosphoryl group, results in elimination of two negativecharges and generates products that have a net positive charge of one.In this experiment, the method of isoelectric focusing (IEF) was used todemonstrate a change from one negative to one positive net charge forthe described substrates during dephosphorylation.

Substrates 70, 74, 75 and 76 were synthesized by standardphosphoramidite chemistries and deprotected for 24 hours at 22° C. in 14M aqueous ammonium hydroxide solution, after which the solvent wasremoved in vacuo. The dried powders were resuspended in 200 μl of H₂Oand filtered through 0.2 μm filters. The concentration of the stocksolutions was estimated by UV-absorbance at 261 nm of samples diluted200-fold in H₂O using a spectrophotometer (Spectronic Genesys 2, MiltonRoy, Rochester, N.Y.).

Dephosphorylation of compounds 70 and 74, 75 and 76 was accomplished bytreating 10 μl of the crude stock solutions (ranging in concentrationfrom approximately 0.5 to 2 mM) with 2 units of CIAP in 100 μl of CIAPbuffer (Promega) at 37° C. for 1 hour. The reactions were then heated to75° C. for 15 min. in order to inactivate the CIAP. For clarity,dephosphorylated compounds are designated ‘dp’. For example, afterdephosphorylation, substrate 70 becomes 70dp.

To prepare samples for EF experiments, the concentration of the stocksolutions of substrate and dephosphorylated product were adjusted to auniform absorbance of 8.5 ×10⁻³ at 532 nm by dilution with water. Twomicroliters of each sample were analyzed by IEF using a PhastSystemelectrophoresis unit (Pharmacia) and PhastGel IEF 3-9 media (Pharmacia)according to the manufacturer's protocol. Separation was performed at15° C. with the following program: pre-run; 2,000 V, 2.5 mA, 3.5 W, 75Vh; load; 200 V, 2.5 mA, 3.5 W, 15 Vh; run; 2,000 V; 2.5 mA; 3.5 W, 130Vh. After separation, samples were visualized by using the FMBIO ImageAnalyzer (Hitachi) fitted with a 585 nm filter. The resulting imagerscan is shown in FIG. 48.

FIG. 48 shows results of IEF separation of substrates 70, 74, 75 and 76and their dephosphorylated products. The arrow labeled “Sample LoadingPosition” indicates a loading line, the ‘+’ sign shows the position ofthe positive electrode and the ‘−’ sign indicates the position of thenegative electrode.

The results shown in FIG. 48 demonstrate that substrates 70, 74, 75 and76 migrated toward the positive electrode, while the dephosphorylatedproducts 70dp, 74dp, 75dp and 76dp migrated toward negative electrode.The observed differences in mobility direction was in accord withpredicted net charge of the substrates (minus one) and the products(plus one). Small perturbations in the mobilities of the phosphorylatedcompounds indicate that the overall pIs vary. This was also true for thedephosphorylated compounds. The presence of the cytosine in 76dp, forinstance, moved this compound further toward the negative electrode,which was indicative of a higher overall pI relative to the otherdephosphorylated compounds. It is important to note that additionalpositive charges can be obtained by using a combination of natural aminomodified bases (70dp and 74dp) along with uncharged methylphosphonatebridges (products 75dp and 76dp).

The results shown above demonstrate that the removal of a singlephosphate group can flip the net charge of an oligonucleotide to causereversal in an electric field, allowing easy separation of products, andthat the precise base composition of the oligonucleotides affectabsolute mobility but not the charge-flipping effect.

Example 23 Detection of Specific Cleavage Products in theINVADER-Directed Cleavage Reaction by Charge Reversal

In this Example the ability to isolate products generated in theINVADER-directed cleavage assay from all other nucleic acids present inthe reaction cocktail was demonstrated using charge reversal. Thisexperiment utilized the following Cy3-labeled oligonucleotide:5′-Cy3-AminoT-AminoT-CTTTTCACCAGCGAGACGGG-3′ (SEQ ID NO:50; termed“oligo 61”). Oligo 61 was designed to release upon cleavage a netpositively charged labeled product. To test whether or not a netpositively charged 5′-end labeled product would be recognized by theCLEAVASE enzymes in the INVADER-directed cleavage assay format, probeoligo 61 (SEQ ID NO:50) and invading oligonucleotide 67 (SEQ ID NO:51)were chemically synthesized on a DNA synthesizer (ABI 391) usingstandard phosphoramidite chemistries and reagents obtained from GlenResearch (Sterling, Va.).

Each assay reaction comprised 100 fmoles of M13mp18 single stranded DNA,10 pmoles each of the probe (SEQ ID NO:50) and INVADER (SEQ ID NO:51)oligonucleotides, and 20 units of CLEAVASE A/G in a 10 μl solution of 10mM MOPS, pH 7.4 with 100 mM KCl. Samples were overlaid with mineral oilto prevent evaporation. The samples were brought to either 50° C., 55°C., 60° C., or 65° C. and cleavage was initiated by the addition of 1 μlof 40 mM MnCl₂. Reactions were allowed to proceed for 25 minutes andthen were terminated by the addition of 10 μl of 95% formamidecontaining 20 mM EDTA and 0.02% methyl violet. The negative controlexperiment lacked the target M13mp18 and was run at 60° C. Fivemicroliters of each reaction were loaded into separate wells of a 20%denaturing polyacrylamide gel (cross-linked 29: 1) with 8 M urea in abuffer containing 45 mM Tris-Borate (pH 8.3) and 1.4 mM EDTA. Anelectric field of 20 watts was applied for 30 minutes, with theelectrodes oriented as indicated in FIG. 49B (i.e., in reverseorientation). The products of these reactions were visualized using theFMBIO fluorescence imager and the resulting imager scan is shown in FIG.49B.

FIG. 49A provides a schematic illustration showing an alignment of theINVADER (SEQ ID NO:50) and probe (SEQ ID NO:51) along the target M13mp18DNA; only 53 bases of the M13mp18 sequence is shown (SEQ ID NO:52). Thesequence of the INVADER oligonucleotide is displayed under the M13mp18target and an arrow is used above the M13mp18 sequence to indicate theposition of the INVADER relative to the probe and target. As shown inFIG. 49A, the INVADER and probe oligonucleotides share a 2 base regionof overlap.

In FIG. 49B, lanes 1-6 contain reactions performed at 50° C., 55° C.,60° C., and 65° C., respectively; lane 5 contained the control reaction(lacking target). In FIG. 49B, the products of cleavage are seen as darkbands in the upper half of the panel; the faint lower band seen appearsin proportion to the amount of primary product produced and, while notlimiting the invention to a particular mechanism, may represent cleavageone nucleotide into the duplex. The uncleaved probe does not enter thegel and is thus not visible. The control lane showed no detectablesignal over background (lane 5). As expected in an invasive cleavagereaction, the rate of accumulation of specific cleavage product wastemperature-dependent. Using these particular oligonucleotides andtarget, the fastest rate of accumulation of product was observed at 55°C. (lane 2) and very little product observed at 65° C. (lane 4).

When incubated for extended periods at high temperature, DNA probes canbreak non-specifically (i.e., suffer thermal degradation) and theresulting fragments contribute an interfering background to theanalysis. The products of such thermal breakdown are distributed fromsingle-nucleotides up to the full length probe. In this experiment, theability of charge based separation of cleavage products (i.e., chargereversal) would allow the sensitive separation of the specific productsof target-dependent cleavage from probe fragments generated by thermaldegradation was examined.

To test the sensitivity limit of this detection method, the targetM13mp18 DNA was serially diluted ten fold over than range of 1 fmole to1 amole. The INVADER and probe oligonucleotides were those describedabove (i.e., SEQ ID NOS:50 and 51). The invasive cleavage reactions wererun as described above with the following modifications: the reactionswere performed at 55° C., 250 mM or 100 mM KGlu was used in place of the100 mM KCl and only 1 pmole of the INVADER oligonucleotide was added.The reactions were initiated as described above and allowed to progressfor 12.5 hours. A negative control reaction that lacked added M13m18target DNA was also run. The reactions were terminated by the additionof 10 μl of 95% formamide containing 20 mM EDTA and 0.02% methyl violet,and 5 μl of these mixtures were electrophoresed and visualized asdescribed above. The resulting imager scan is shown in FIG. 50.

In FIG. 50, lane 1 contains the negative control; lanes 2-5 containreactions performed using 100 mM KGlu; lanes 6-9 contain reactionsperformed using 250 mM KGlu. The reactions resolved in lanes 2 and 6contained 1 fmole of target DNA; those in lanes 3 and 7 contained 100amole of target; those in lanes 4 and 8 contained 10 amole of target andthose in lanes 5 and 9 contained 1 amole of target. The results shown inFIG. 50 demonstrate that the detection limit using charge reversal todetect the production of specific cleavage products in an invasivecleavage reaction is at or below 1 attomole or approximately 6.02×10⁵target molecules. No detectable signal was observed in the control lane,which indicates that non-specific hydrolysis or other breakdown productsdo not migrate in the same direction as enzyme-specific cleavageproducts. The excitation and emission maxima for Cy3 are 554 and 568,respectively, while the FMBIO Imager Analyzer excites at 532 and detectsat 585. Therefore, the limit of detection of specific cleavage productscan be improved by the use of more closely matched excitation source anddetection filters.

Example 24 Devices and Methods for the Separation and Detection ofCharged Reaction Products

This Example is directed at methods and devices for isolating andconcentrating specific reaction products produced by enzymatic reactionsconducted in solution whereby the reactions generate charged productsfrom either a charge neutral substrate or a substrate bearing theopposite charge borne by the specific reaction product. The methods anddevices of this Example allow isolation of, for example, the productsgenerated by the INVADER-directed cleavage assay of the presentinvention.

The methods and devices of this Example are based on the principle thatwhen an electric field is applied to a solution of charged molecules,the migration of the molecules toward the electrode of the oppositecharge occurs very rapidly. If a matrix or other inhibitory material isintroduced between the charged molecules and the electrode of oppositecharge such that this rapid migration is dramatically slowed, the firstmolecules to reach the matrix will be nearly stopped, thus allowing thelagging molecules to catch up. In this way a dispersed population ofcharged molecules in solution can be effectively concentrated into asmaller volume. By tagging the molecules with a detectable moiety (e.g.,a fluorescent dye), detection is facilitated by both the concentrationand the localization of the analytes. This Example illustrates twoembodiments of devices contemplated by the present invention; of course,variations of these devices will be apparent to those skilled in the artand are within the spirit and scope of the present invention.

FIG. 51 depicts one embodiment of a device for concentrating thepositively-charged products generated using the methods of the presentinvention. As shown in FIG. 51, the device comprises a reaction tube(10) that contains the reaction solution (11). One end of each of twothin capillaries (or other tubes with a hollow core) (13A and 13B) aresubmerged in the reaction solution (11). The capillaries (13A and 13B)may be suspended in the reaction solution (11) such that they are not incontact with the reaction tube itself; one appropriate method ofsuspending the capillaries is to hold them in place with clamps (notshown). Alternatively, the capillaries may be suspended in the reactionsolution (11) such that they are in contact with the reaction tubeitself. Suitable capillaries include glass capillary tubes commonlyavailable from scientific supply companies (e.g., Fisher Scientific orVWR Scientific) or from medical supply houses that carry materials forblood drawing and analysis. Though the present invention is not limitedto capillaries of any particular inner diameter, tubes with innerdiameters of up to about ⅛ inch (approximately 3 mm) are particularlypreferred for use with the present invention; for example, Kimble No.73811-99 tubes (VWR Scientific) have an inner diameter of 1.1 mm and area suitable type of capillary tube. Although the capillaries of thedevice are commonly composed of glass, any nonconductive tubularmaterial, either rigid or flexible, that can contain either a conductivematerial or a trapping material is suitable for use in the presentinvention. One example of a suitable flexible tube is Tygon® clearplastic tubing (Part No. R3603; inner diameter= 1/16 inch; outerdiameter=⅛ inch).

As illustrated in FIG. 51, capillary 13A is connected to the positiveelectrode of a power supply (20) (e.g., a controllable power supplyavailable through the laboratory suppliers listed above or throughelectronics supply houses like Radio Shack) and capillary 13B isconnected to the negative electrode of the power supply (20). Capillary13B is filled with a trapping material (14) capable of trapping thepositively-charged reaction products by allowing minimal migration ofproducts that have entered the trapping material (14). Suitable trappingmaterials include, but are not limited to, high percentage (e.g., about20%) acrylamide polymerized in a high salt buffer (0.5 M or highersodium acetate or similar salt); such a high percentage polyacrylamidematrix dramatically slows the migration of the positively-chargedreaction products. Alternatively, the trapping material may comprise asolid, negatively-charged matrix, such as negatively-charged latexbeads, that can bind the incoming positively-charged products. It shouldbe noted that any amount of trapping material (14) capable of inhibitingany concentrating the positively-charged reaction products may be used.Thus, while the capillary 13B in FIG. 51 only contains trapping materialin the lower, submerged portion of the tube, the trapping material (14)can be present in the entire capillary (13B); similarly, less trappingmaterial (14) could be present than that shown in FIG. 51 because thepositively-charged reaction products generally accumulate within a verysmall portion of the bottom of the capillary (13B). The amount oftrapping material need only be sufficient to make contact with thereaction solution (11) and have the capacity to collect the reactionproducts. When capillary 13B is not completely filled with the trappingmaterial, the remaining space is filled with any conductive material(15); suitable conductive materials are discussed below.

By comparison, the capillary (13A) connected to the positive electrodeof the power supply 20 may be filled with any conductive material (15;indicated by the hatched lines in FIG. 51). This may be the samplereaction buffer (e.g., 10 mM MOPS, pH 7.5 with 150 mM LiCl, 4 mM MnCl₂),a standard electrophoresis buffer (e.g., 45 mM Tris-Borate, pH 8.3, 1.4mM EDTA), or the reaction solution (11) itself. The conductive material(15) is frequently a liquid, but a semi-solid material (e.g., a gel) orother suitable material might be easier to use and is within the scopeof the present invention. Moreover, that trapping material used in theother capillary (i.e., capillary 13B) may also be used as the conductivematerial. Conversely, it should be noted that the same conductivematerial used in the capillary (13A) attached to the positive electrodemay also be used in capillary 13B to fill the space above the regioncontaining the trapping material (14) (see FIG. 51).

The top end of each of the capillaries (13A and 13B) is connected to theappropriate electrode of the power supply (20) by electrode wire (18) orother suitable material. Fine platinum wire (e.g., 0.1 to 0.4 mm, AesarJohnson Matthey, Ward Hill, Mass.) is commonly used as conductive wirebecause it does not corrode under electrophoresis conditions. Theelectrode wire (18) can be attached to the capillaries (13A and 13B) bya nonconductive adhesive (not shown), such as the silicone adhesivesthat are commonly sold in hardware stores for sealing plumbing fixtures.If the capillaries are constructed of a flexible material, the electrodewire (18) can be secured with a small hose clamp or constricting wire(not shown) to compress the opening of the capillaries around theelectrode wire. If the conducting material (15) is a gel, an electrodewire (18) can be embedded directly in the gel within the capillary.

The cleavage reaction is assembled in the reaction tube (10) and allowedto proceed therein as described in proceeding Examples (e.g., Examples22-23). Though not limited to any particular volume of reaction solution(11), a preferred volume is less than 10 ml and more preferably lessthan 0.1 ml. The volume need only be sufficient to permit contact withboth capillaries. After the cleavage reaction is completed, an electricfield is applied to the capillaries by turning on the power source (20).As a result, the positively-charged products generated in the course ofthe INVADER-directed cleavage reaction that employs an oligonucleotide,which when cleaved, generates a positively charged fragment (describedin Ex. 23) but when uncleaved bears a net negative charge, migrate tothe negative capillary, where their migration is slowed or stopped bythe trapping material (14), and the negatively-charged uncut andthermally degraded probe molecules migrate toward the positiveelectrode. Through the use of this or a similar device, thepositively-charged products of the invasive cleavage reaction areseparated from the other material (i.e., uncut and thermally degradedprobe) and concentrated from a large volume. Concentration of theproduct in a small amount of trapping material (14) allows forsimplicity of detection, with a much higher signal-to-noise ratio thanpossible with detection in the original reaction volume. Because theconcentrated product is labeled with a detectable moiety like afluorescent dye, a commercially-available fluorescent plate reader (notshown) can be used to ascertain the amount of product. Suitable platereaders include both top and bottom laser readers. Capillary 13B can bepositioned with the reaction tube (10) at any desired position so as toaccommodate use with either a top or a bottom plate reading device.

In the alternative embodiment of the present invention depicted in FIG.52, the procedure described above is accomplished by utilizing only asingle capillary (13B). The capillary (13B) contains the trappingmaterial (14) described above and is connected to an electrode wire(18), which in turn is attached to the negative electrode of a powersupply (20). The reaction tube (10) has an electrode (25) embedded intoits surface such that one surface of the electrode is exposed to theinterior of the reaction tube (10) and another surface is exposed to theexterior of the reaction tube. The surface of the electrode (25) on theexterior of the reaction tube is in contact with a conductive surface(26) connected to the positive electrode of the power supply (20)through an electrode wire (18). Variations of the arrangement depictedin FIG. 52 are also contemplated by the present invention. For example,the electrode (25) may be in contact with the reaction solution (11)through the use of a small hole in the reaction tube (10); furthermore,the electrode wire (18) can be directly attached to the electrode wire(18), thereby eliminating the conductive surface (26).

As indicated in FIG. 52, the electrode (25) is embedded in the bottom ofa reaction tube (10) such that one or more reaction tubes may be set onthe conductive surface (26). This conductive surface could serve as anegative electrode for multiple reaction tubes; such a surface withappropriate contacts could be applied through the use of metal foils(e.g., copper or platinum, Aesar Johnson Matthey, Ward Hill, Mass.) inmuch the same way contacts are applied to circuit boards. Because such asurface contact would not be exposed to the reaction sample directly,less expensive metals, such as the copper could be used to make theelectrical connections.

The above devices and methods are not limited to separation andconcentration of positively charged oligonucleotides. As will beapparent to those skilled in the art, negatively charged reactionproducts may be separated from neutral or positively charged reactantsusing the above device and methods with the exception that capillary 13Bis attached to the positive electrode of the power supply (20) andcapillary 13A or alternatively, electrode 25, is attached to thenegative electrode of the power supply (20).

Example 25 Primer-Directed and Primer Independent Cleavage Occur at theSame Site when the Primer Extends to the 3′ Side of a Mismatched“Bubble” in the Downstream Duplex

As discussed above in Example 1, the presence of a primer upstream of abifurcated duplex can influence the site of cleavage, and the existenceof a gap between the 3′ end of the primer and the base of the duplex cancause a shift of the cleavage site up the unpaired 5′ arm of thestructure (see also Lyamichev et al., supra and U.S. Pat. No.5,422,253). The resulting non-invasive shift of the cleavage site inresponse to a primer is demonstrated in FIGS. 8, 9 and 10, in which theprimer used left a 4-nucleotide gap (relative to the base of theduplex). In FIGS. 8-10, all of the “primer-directed” cleavage reactionsyielded a 21 nucleotide product, while the primer-independent cleavagereactions yielded a 25 nucleotide product. The site of cleavage obtainedwhen the primer was extended to the base of the duplex, leaving no gapwas examined. The results are shown in FIG. 53 (FIG. 53 is areproduction of FIG. 2C in Lyamichev et al. These data were derived fromthe cleavage of the structure shown in FIG. 5, as described inExample 1. Unless otherwise specified, the cleavage reactions comprised0.01 pmoles of heat-denatured, end-labeled hairpin DNA (with theunlabeled complementary strand also present), 1 pmole primer(complementary to the 3′ arm shown in FIG. 5 and having the sequence:5′-GAATTCGATTTAGGTGACAC TATAGAATACA [SEQ ID NO:53]) and 0.5 units ofDNAPTaq (estimated to be 0.026 pmoles) in a total volume of 10 μl of 10mM Tris-Cl, pH 8.5, and 1.5 mM MgCl₂ and 50 mM KCl. The primer wasomitted from the reaction shown in the first lane of FIG. 53 andincluded in lane 2. These reactions were incubated at 55° C. for 10minutes. Reactions were initiated at the final reaction temperature bythe addition of either the MgCl₂ or enzyme. Reactions were stopped attheir incubation temperatures by the addition of 8 μl of 95% formamidewith 20 mM EDTA and 0.05% marker dyes.

FIG. 53 is an autoradiogram that indicates the effects on the site ofcleavage of a bifurcated duplex structure in the presence of a primerthat extends to the base of the hairpin duplex. The size of the releasedcleavage product is shown to the left (i.e., 25 nucleotides). Adideoxynucleotide sequencing ladder of the cleavage substrate is shownon the right as a marker (lanes 3-6).

These data show that the presence of a primer that is adjacent to adownstream duplex (lane 2) produces cleavage at the same site as seen inreactions performed in the absence of the primer (lane 1). (See FIGS. 8Aand B, 9B and 10A for additional comparisons). When the 3′ terminalnucleotides of the upstream oligonucleotide can base pair to thetemplate strand but are not homologous to the displaced strand in theregion immediately upstream of the cleavage site (i.e., when theupstream oligonucleotide is opening up a “bubble” in the duplex), thesite to which cleavage is apparently shifted is not wholly dependent onthe presence of an upstream oligonucleotide.

As discussed above in the Background, and in Table 1, the requirementthat two independent sequences be recognized in an assay provides ahighly desirable level of specificity. In the invasive cleavagereactions of the present invention, the INVADER and probeoligonucleotides must hybridize to the target nucleic acid with thecorrect orientation and spacing to enable the production of the correctcleavage product. When the distinctive pattern of cleavage is notdependent on the successful alignment of both oligonucleotides in thedetection system these advantages of independent recognition are lost.

Example 26 Invasive Cleavage and Primer-Directed Cleavage when there isOnly Partial Homology in the “X” Overlap Region

While not limiting the present invention to any particular mechanism,invasive cleavage occurs when the site of cleavage is shifted to a sitewithin the duplex formed between the probe and the target nucleic acidin a manner that is dependent on the presence of an upstreamoligonucleotide that shares a region of overlap with the downstreamprobe oligonucleotide. In some instances, the 5′ region of thedownstream oligonucleotide may not be completely complementary to thetarget nucleic acid. In these instances, cleavage of the probe may occurat an internal site within the probe even in the absence of an upstreamoligonucleotide (in contrast to the base-by-base nibbling seen when afully paired probe is used without an INVADER). Invasive cleavage ischaracterized by an apparent shifting of cleavage to a site within adownstream duplex that is dependent on the presence of the INVADERoligonucleotide.

A comparison between invasive cleavage and primer-directed cleavage maybe illustrated by comparing the expected cleavage sites of a set ofprobe oligonucleotides having decreasing degrees of complementarity tothe target strand in the 5′ region of the probe (i.e., the region thatoverlaps with the INVADER). A simple test, similar to that performed onthe hairpin substrate above (Ex. 25), can be performed to compareinvasive cleavage with the non- invasive primer-directed cleavagedescribed above. Such a set of test oligonucleotides is diagrammed inFIG. 54. The structures shown in FIG. 54 are grouped in pairs, labeled“a”, “b”, “c”, and “d”. Each pair has the same probe sequence annealedto the target strand (SEQ ID NO:54), but the top structure of each pairis drawn without an upstream oligonucleotide, while the bottom structureincludes this oligonucleotide (SEQ ID NO:55). The sequences of theprobes shown in FIGS. 54 a-54 d are listed in SEQ ID NOS:32, 56, 57 and58, respectively. Probable sites of cleavage are indicated by the blackarrowheads. (It is noted that the precise site of cleavage on each ofthese structures may vary depending on the choice of cleavage agent andother experimental variables. These particular sites are provided forillustrative purposes only.) To conduct this test, the site of cleavageof each probe is determined both in the presence and the absence of theupstream oligonucleotide, in reaction conditions such as those describedin Example 18. The products of each pair of reactions are then becompared to determine whether the fragment released from the 5′ end ofthe probe increases in size when the upstream oligonucleotide isincluded in the reaction.

The arrangement shown in FIG. 54 a, in which the probe molecule iscompletely complementary to the target strand, is similar to that shownin FIG. 28. Treatment of the top structure with the 5′ nuclease of a DNApolymerase would cause exonucleolytic nibbling of the probe (i.e., inthe absence of the upstream oligonucleotide). In contrast, inclusion ofan INVADER oligonucleotide would cause a distinctive cleavage shiftsimilar, to those observed in FIG. 29.

The arrangements shown in FIGS. 54 b and 54 c have some amount ofunpaired sequence at the 5′ terminus of the probe ( 3 and 5 bases,respectively). These small 5′arms are suitable cleavage substrate forthe 5′ nucleases and would be cleaved within 2 nucleotide's of thejunction between the single stranded region and the duplex. In thesearrangements, the 3′ end of the upstream oligonucleotide shares identitywith a portion of the 5′ region of the probe that is complementary tothe target sequence (that is the 3′ end of the INVADER has to competefor binding to the target with a portion of the 5′ end of the probe).Therefore, when the upstream oligonucleotide is included it is thoughtto mediate a shift in the site of cleavage into the downstream duplex(although the present invention is not limited to any particularmechanism of action), and this would, therefore, constitute invasivecleavage. If the extreme 5′ nucleotides of the unpaired region of theprobe were able to hybridize to the target strand, the cleavage site inthe absence of the INVADER might change but the addition of the INVADERoligonucleotide would still shift the cleavage site to the properposition.

Finally, in the arrangement shown in FIG. 54 d, the probe and upstreamoligonucleotides share no significant regions of homology, and thepresence of the upstream oligonucleotide would not compete for bindingto the target with the probe. Cleavage of the structures shown in FIG.54 d would occur at the same site with or without the upstreamoligonucleotide, and is thus would not constitute invasive cleavage.

By examining any upstream oligonucleotide/probe pair in this way, it caneasily be determined whether the resulting cleavage is invasive ormerely primer-directed. Such analysis is particularly useful when theprobe is not fully complementary to the target nucleic acid, so that theexpected result may not be obvious by simple inspection of thesequences.

Example 27 Modified CLEAVASE Enzymes

In order to develop nucleases having useful activities for the cleavageof nucleic acids the following modified nucleases were produced.

a) CLEAVASE BN/thrombin Nuclease

i) Cloning and Expression of CLEAVASE BN/thrombin Nuclease

Site directed mutagenesis was used to introduce a protein sequencerecognized by the protease thrombin into the region of the CLEAVASE BNnuclease that is thought to form the helical arch of the protein throughwhich the single-stranded DNA that is cleaved must presumably pass.Mutagenesis was carried out using the Transformer™ mutagenesis kit(Clonetech, Palo Alto, Calif.) according to manufacturer's protocolusing the mutagenic oligonucleotide 5′-GGGAAAGTCCTCGCAGCCGCGCGGGACGAGCGTGGGGGCCCG (SEQ ID NO:59). After mutagenesis, the DNA wassequenced to verify the insertion of the thrombin cleavage site. The DNAsequence encoding the CLEAVASE BN/thrombin nuclease is provided in SEQID NO:60; the amino acid sequence of CLEAVASE BN/thrombin nuclease isprovided in SEQ ID NO:61.

A large scale preparation of the thrombin mutant (i.e., CLEAVASEBN/thrombin) was done using E. coli cells overexpressing the CLEAVASEBN/thrombin nuclease as described in Example 28.

ii) Thrombin Cleavage of CLEAVASE BN/thrombin

Six point four (6.4) mg of the purified CLEAVASE BN/thrombin nucleasewas digested with 0.4 U of thrombin (Novagen) for 4 hours at 23° C. or37° C. Complete digestion was verified by electrophoresis on a 15% SDSpolyacrylamide gel followed by staining with Coomassie Brilliant Blue R.Wild-type CLEAVASE BN nuclease was also digested with thrombin as acontrol. The resulting gel is shown in FIG. 61.

In FIG. 61, lane 1 contains molecular weight markers (Low-Range ProteinMolecular Weight Markers; Promega), lane 2 contains undigested CLEAVASEBN/throbin nuclease, lanes 3 and 4 contain CLEAVASE BN/thrombin nucleasedigested with thrombin at 23° C. for 2 and 4 hours, respectively, andlanes 5 and 6 contain CLEAVASE BN/thrombin nuclease digested withthrombin at 37° C. for 2 and 4 hours, respectively. These results showthat the CLEAVASE BN/thrombin nuclease has an apparent molecular weightof 36.5 kilodaltons and demonstrate that CLEAVASE BN/thrombin nucleaseis efficiently cleaved by thrombin. In addition, the thrombin cleavageproducts have approximate molecular weights of 27 kilodaltons and 9kilodaltons, the size expected based upon the position of the insertedthrombin site in the CLEAVASE BN/thrombin nuclease.

To determine the level of hairpin cleavage activity in digested andundigested CLEAVASE BN/thrombin nuclease, dilutions were made and usedto cleave a test hairpin containing a 5′ fluoroscein label. Varyingamounts of digested and undigested CLEAVASE BN/thrombin nuclease wereincubated with 5 μM oligonucleotide S-60 hairpin (SEQ ID NO:29; see FIG.26) in 10 mM MOPS (pH 7.5), 0.05% Tween-20, 0.05% NP-40, and I mM MnCl₂for 5 minutes at 60° C. The digested mixture was electrophoresed on a20% acrylamide gel and visualized on a Hitachi FMBIO 100 fluoroimager.The resulting image is shown in FIG. 62.

In FIG. 62, lane 1 contains the no enzyme control, lane 2 containsreaction products produced using 0.01 ng of CLEAVASE BN nuclease, lanes3, 4, and 5 contain reaction products produced using 0.01 ng, 0.04 ng,and 4 ng of undigested CLEAVASE BN/thrombin nuclease, respectively, andlanes 6, 7, and 8 contain reaction products produced using 0.01 ng, 0.04ng, and 4 ng of thrombin-digested CLEAVASE BN/thrombin nuclease,respectively. The results shown in FIG. 62 demonstrated that theinsertion of the thrombin cleavage site reduced cleavage activity about200-fold (relative to the activity of CLEAVASE BN nuclease), but thatdigestion with thrombin did not reduce the activity significantly.

M13 single-stranded DNA was used as a substrate for cleavage by CLEAVASEBN nuclease and digested and undigested CLEAVASE BN/thrombin nuclease.Seventy nanograms of single-stranded M13 DNA (NEB) was incubated in 10mM MOPS, pH 7.5, 0.05% Tween-20, 0.05% NP-40, 1 mM MgCl₂ or 1 mM MnCl₂,with 8 ng of CLEAVASE BN nuclease, undigested CLEAVASE BN/thrombinnuclease, or digested CLEAVASE BN/thrombin nuclease for 10 minutes at50° C. Reaction mixtures were electrophoresed on a 0.8% agarose gel andthen stained with a solution containing 0.5 μg/ml ethidium bromide(EtBr) to visualize DNA bands. A negative image of the EtBr-stained gelis shown in FIG. 63.

In FIG. 63, lane 1 contains the no enzyme control, lane 2 containsreaction products produced using CLEAVASE BN nuclease and 1 mM MgCl₂,lane 3 contains reaction products produced using CLEAVASE BN nucleaseand 1 mM MnCl₂, lane 4 contains reaction products produced usingundigested CLEAVASE BN/thrombin nuclease and 1 mM MgCl₂, lane 5 containsreaction products produced using undigested CLEAVASE BN/thrombinnuclease and 1 mM MnCl₂, lane 6 contains reaction products producedusing thrombin-digested CLEAVASE BN/thrombin nuclease and 1 mM MgCl₂,and lane 7 contains reaction products produced using thrombin-digestedCLEAVASE BN/thrombin nuclease and 1 mM MnCl₂. The results shown in FIG.63 demonstrated that the CLEAVASE BN/thrombin nuclease had an enhancedability to cleave circular DNA (and thus a reduced requirement for thepresence of a free 5′ end) as compared to the CLEAVASE BN nuclease.

It can be seen from these data that the helical arch of these proteinscan be opened without destroying the enzyme or its ability tospecifically recognize cleavage structures. The CLEAVASE BN/thrombinmutant has an increased ability to cleave without reference to a 5′ end,as discussed above. The ability to cleave such structures will allow thecleavage of long molecules, such as genomic DNA that, while often notcircular, may present many desirable cleavage sites that are at a farremoved from any available 5′ end. Cleavage structures may be made atsuch sites either by folding of the strands (i.e., CFLP® cleavage) or bythe introduction of structure-forming oligonucleotides (U.S. Pat. No.5,422,253). 5′ ends of nucleic acids can also be made unavailablebecause of binding of a substance too large to thread through thehelical arch. Such binding moieties may include proteins such asstreptavidin or antibodies, or solid supports such as beads or the wallsof a reaction vessel. A cleavage enzyme with an opening in the loop ofthe helical arch will be able to cleave DNAs that are configured in thisway, extending the number of ways in which reactions using such enzymescan be formatted.

b) CLEAVASE DN Nuclease

i) Construction and Expression of CLEAVASE DN Nuclease

A polymerization deficient mutant of Taq DNA polymerase, termed CLEAVASEDN nuclease, was constructed. CLEAVASE DN nuclease contains anasparagine residue in place of the wild-type aspartic acid residue atposition 785 (D785N).

DNA encoding the CLEAVASE DN nuclease was constructed from the geneencoding for CLEAVASE A/G (mutTaq, Ex. 2) in two rounds of site-directedmutagenesis. First, the G at position 1397 and the G at position 2264 ofthe CLEAVASE A/G gene (SEQ ID NO:21) were changed to A at each positionto recreate a wild-type DNAPTaq gene. As a second round of mutagenesis,the wild type DNAPTaq gene was converted to the CLEAVASE DN gene bychanging the G at position 2356 to A. These manipulations were performedas follows.

DNA encoding the CLEAVASE A/G nuclease was recloned from pTTQ18 plasmid(Ex. 2) into the pTrc99A plasmid (Pharmacia) in a two step procedure.First, the pTrc99A vector was modified by removing the G at position 270of the pTrc99A map, creating the pTrc99G cloning vector. To this end,pTrc99A plasmid DNA was cut with NcoI and the recessive 3′ ends werefilled-in using the Klenow fragment of E.coli polymerase I in thepresence of all four dNTPs at 37° C. for 15 min. After inactivation ofthe Klenow fragment by incubation at 65° C. for 10 min, the plasmid DNAwas cut with EcoRI, the ends were again filled-in using the Klenowfragment in the presence of all four dNTPs at 37° C. for 15 min. TheKlenow fragment was then inactivated by incubation at 65° C. for 10 min.The plasmid DNA was ethanol precipitated, recircularized by ligation,and used to transform E.coli JM109 cells (Promega). Plasmid DNA wasisolated from single colonies and deletion of the G at position 270 ofthe pTrc99A map was confirmed by DNA sequencing.

As a second step, DNA encoding the CLEAVASE A/G nuclease was removedfrom the pTTQ 18 plasmid using EcoRI and SalI and the DNA fragmentcarrying the CLEAVASE A/G nuclease gene was separated on a 1% agarosegel and isolated with Geneclean II Kit (Bio 101, Vista, Calif.). Thepurified fragment was ligated into the pTrc99G vector, which had beencut with EcoRI and SalI. The ligation mixture was used to transformcompetent E.coli JM1O9 cells (Promega). Plasmid DNA was isolated fromsingle colonies and insertion of the CLEAVASE A/G nuclease gene wasconfirmed by restriction analysis using EcoRI and SalI.

Plasmid DNA pTrcAG carrying the CLEAVASE A/G nuclease gene cloned intothe pTrc99A vector was purified from 200 ml of JM109 overnight cultureusing QIAGEN Plasmid Maxi kit (QIAGEN, Chatsworth,Calif.) according tomanufacturer's protocol. pTrcAG plasmid DNA was mutagenized using twomutagenic primers, E465 (SEQ ID NO:62) (Integrated DNA Technologies,Iowa) and R754Q (SEQ ID NO:63) (Integrated DNA Technologies), and theselection primer Trans Oligo AlwNI/SpeI (Clontech, Palo Alto, Calif.,catalog #6488-1) according to Transformer™ Site-Directed Mutagenesis Kitprotocol (Clontech) to produce a restored wild-type DNAPTaq gene(pTrcWT).

pTrcWT plasmid DNA carrying the wild-type DNAPTaq gene cloned into thepTrc99A vector was purified from 200 ml of JM109 overnight culture usingQIAGEN Plasmid Maxi kit (QIAGEN, Chatsworth, Calif.) according tomanufacturer's protocol. pTrcWT was then mutagenized using the mutagenicprimer D785N (SEQ ID NO:64) (Integrated DNA Technologies) and theselection primer Switch Oligo SpeI/AlwNI (Clontech, catalog #6373-1)according to Transformer™ Site-Directed Mutagenesis Kit protocol(Clontech) to create a plasmid containing DNA encoding the CLEAVASE DNnuclease. The DNA sequence encoding the CLEAVASE DN nuclease is providedin SEQ ID NO:65; the amino acid sequence of CLEAVASE DN nuclease isprovided in SEQ ID NO:66.

A large scale preparation of the CLEAVASE DN nuclease was done using E.coli cells overexpressing the CLEAVASE DN nuclease as described inExample 28.

c) CLEAVASE DA Nuclease and CLEAVASE DV Nuclease

Two polymerization deficient mutants of Taq DNA polymerase, termedCLEAVASE DA nuclease and CLEAVASE DV nuclease, were constructed. TheCLEAVASE DA nuclease contains a alanine residue in place of thewild-type aspartic acid residue at position 610 (D785A). The CLEAVASE DVnuclease contains a valine residue in place of the wild-type asparticacid residue at position 610 (D610V).

i) Construction and Expression of the CLEAVASE DA and CLEAVASE DVNucleases

To construct vectors encoding the CLEAVASE DA and DV nucleases, theCLEAVASE A/G nuclease gene contained within pTrcAG was mutagenized withtwo mutagenic primers, R754Q (SEQ ID NO:63) and D610AV (SEQ ID NO:67)and the selection primer Trans Oligo AlwNI/SpeI (Clontech, catalog#6488-1) according to the Transformer™ Site-Directed Mutagenesis Kitprotocol (Clontech,) to create a plasmid containing DNA encoding theCLEAVASE DA nuclease or CLEAVASE DV nuclease. The D610AV oligonucleotidewas synthesized to have a purine, A or G, at position 10 from the 5′ endof the oligonucleotide. Following mutagenesis, plasmid DNA was isolatedfrom single colonies and the type of mutation present, DA or DV, wasdetermined by DNA sequencing. The DNA sequence encoding the CLEAVASE DAnuclease is provided in SEQ ID NO:68; the amino acid sequence ofCLEAVASE DA nuclease is provided in SEQ ID NO:69. The DNA sequenceencoding the CLEAVASE DV nuclease is provided in SEQ ID NO:70; the aminoacid sequence of CLEAVASE DV nuclease is provided in SEQ ID NO:71.

d) CLEAVASE Tth DN Nuclease

i) Construction and Expression of CLEAVASE TthDN Nuclease

The DNA polymerase enzyme from the bacterial species Thermusthermophilus (Tth) was produced by cloning the gene for this proteininto an expression vector and overproducing it in E. coli cells. GenomicDNA was prepared from 1 vial of dried Thermus thermophilus strain HB-8from ATCC (ATCC #27634) as described in Ex. 28a. The DNA polymerase genewas amplified by PCR as described in Ex. 27b using the followingprimers: 5′-CACGAATTCCGAGGCGATGCTTCCGCTC-3′ (SEQ ID NO:254) and5′-TCGACGTCGACTAACCCTTGGCGGAAAGCC-3′ (SEQ ID NO:255),as described in Ex.28a.

The resulting PCR product was digested with EcoR I and SalI restrictionendonucleases and inserted into EcoRI/Sal I digested plasmid vectorpTrc99g (described in Example 27b) by ligation, as described in Example27b, to create the plasmid pTrcTth-1. This Tth polymerase construct ismissing a single nucleotide which was inadvertently omitted from the 5′oligonucleotide, resulting in the polymerase gene being out of frame.This mistake was corrected by mutagenesis of pTrcTth-1 as described inExample 27b using the following oligonucleotide:5′-GCATCGCCTCGGAATTCATGGTC-3′ (SEQ ID NO:256), to create the plasmidpTrcTth-2. The Tth DN construct was created by mutating the sequenceencoding an aspartic acid at position 787 to a sequence encodingasparagine. Mutagenesis of pTrcTth-2 with the following oligonucleotide:5′-CAGGAGGAGCTCGTTGTGGACCTGGA-3′ (SEQ ID NO:257) as described in Example27b, to create the plasmid pTrcTth-DN. The resultingpolymerase-deficient nuclease, Cleavase® TthDN was expressed andpurified as described in Ex. 28.

Large scale preparations of the CLEAVASE DA and CLEAVASE DV nucleaseswas done using E. coli cells overexpressing the CLEAVASE DA nuclease orthe CLEAVASE DV nuclease as described in Example 28.

Example 28 Cloning And Expression of Thermostable FEN-1 Endonucleases

Sequences encoding thermostable FEN-1 proteins derived from severalArchaebacterial species were cloned and overexpressed in E. coli . ThisExample involved a) cloning and expression of a FEN-1 endonuclease fromMethanococcus jannaschii; b) cloning and expression of a FEN-1endonuclease from Pyrococcus furiosus; c) cloning and expression of aFEN-1 endonuclease from Pyrococcus woesei; d) cloning and expression ofa FEN-1 endonuclease from Archaeoglobus fulgidus; e) large scalepreparation of recombinant thermostable FEN-1 proteins; and f) activityassays using FEN-1 endonucleases.

a) Cloning and Expression of a FEN-1 Endonuclease from Methanococcusjannaschii

DNA encoding the FEN-1 endonuclease from Methanococcus jannaschii (M.jannaschii) was isolated from M. jannaschii cells and inserted into aplasmid under the transcriptional control of an inducible promoter asfollows. Genomic DNA was prepared from 1 vial of live M. jannaschiibacteria (DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen,Braunschweig, Germany # 2661) with the DNA XTRAX kit (Gull Laboratories,Salt Lake City, Utah) according to the manufacturer's protocol. Thefinal DNA pellet was resuspended in 100 μl of TE (10 mM Tris HCl, pH8.0, 1 mM EDTA). One microliter of the DNA solution was employed in aPCR using the Advantage™ cDNA PCR kit (Clonetech); the PCR was conductedaccording to manufacturer's recommendations. The 5′-end primer (SEQ IDNO:72) is complementary to the 5′ end of the Mja FEN-1 open readingframe with a one base substitution to create an NcoI restriction site (afragment of the M. jannaschii genome that contains the gene encoding M.jannaschii (Mja) FEN-1 is available from GenBank as accession # U67585).The 3′-end primer (SEQ ID NO:73) is complementary to a sequence about 15base pairs downstream from the 3′ end of the Mja FEN-1 open readingframe with 2 base substitutions to create a SalI restriction enzymesite. The sequences of the 5′-end and 3′-end primers are: 5′-GGGATACCATGGGAGTGCAGTTTGG-3′ (SEQ ID NO:72) and 5′-GGTAAATTTTTCTCGTCGACATCCCAC-3′ (SEQ ID NO:73), respectively. The PCR reaction resulted inthe amplification (i.e., production) of a single major band about 1kilobase in length. The open reading frame (ORF) encoding the Mja FEN-1endonuclease is provided in SEQ ID NO:74; the amino acid sequenceencoded by this ORF is provided in SEQ ID NO:75.

Following the PCR amplification, the entire reaction was electrophoresedon a 1.0% agarose gel and the major band was excised from the gel andpurified using the Geneclean II kit (Bio101, Vista, Calif.) according tomanufacturer's instructions. Approximately 1 μg of the gel-purified MjaFEN-1 PCR product was digested with NcoI and SalI. After digestion, theDNA was purified using the Geneclean II kit according to manufacturer'sinstructions. One microgram of the pTrc99a vector (Pharmacia) wasdigested with NcoI and SalI in preparation for ligation with thedigested PCR product. One hundred nanograms of digested pTrc99a vectorand 250 ng of digested Mja FEN-1 PCR product were combined and ligatedto create pTrc99-MJFEN1. pTrc99-MJFEN1 was used to transform competentE. coli JM109 cells (Promega) using standard techniques.

b) Cloning and Expression of a FEN-1 Endonuclease from Pyrococcusfuriosus

DNA encoding the Pyrococcus furiosus (P. furiosus) FEN-1 endonucleasewas obtained by PCR amplification using a plasmid containing DNAencoding the P. furiosus (Pfu) FEN-1 endonuclease (obtained from Dr.Frank Robb, Center of Marine Biotechnology, Baltimore, Md.). DNAsequences encoding a portion of the Pfu FEN-1 endonuclease can beobtained from GenBank as accession Nos. AA113505 and W36094. Theamplified Pfu FEN-1 gene was inserted into the pTrc99a expression vector(Pharmacia) to place the Pfu FEN-1 gene under the transcriptionalcontrol of the inducible trc promoter. The PCR amplification wasconducted as follows. One hundred microliter reactions contained 50 mMTris HCl, pH 9.0, 20 mM (NH₄)₂SO₄, 2 mM MgCl₂, 50 μM dNTPs, 50 pmoleeach primer, 1 U Tfl polymerase (Epicentre Technologies, Madison, Wis.)and 1 ng of FEN-1 gene-containing plasmid DNA. The 5′-end primer (SEQ IDNO:76) is complementary to the 5′ end of the Pfu FEN-1 open readingframe but with two substitutions to create an NcoI site and the 3′-endprimer (SEQ ID NO:77) is complementary to a region located about 30 basepairs downstream of the FEN-1 open reading frame with two substitutionsto create a PstI site. The sequences of the 5′-end and 3′-end primersare: 5′-GAGGTGATACCATG GGTGTCC-3′(SEQ ID NO:76) and5′-GAAACTCTGCAGCGCGTCAG-3′ (SEQ ID NO:77), respectively. The PCRreaction resulted in the amplification of a single major band about 1kilobase in length. The open reading frame (ORF) encoding the Pfu FEN-1endonuclease is provided in SEQ ID NO:78; the amino acid sequenceencoded by this ORF is provided in SEQ ID NO:79.

Following the PCR amplification, the entire reaction was electrophoresedon a 1.0% agarose gel and the major band was excised from the gel andpurified using the Geneclean II kit (Bio101) according to manufacturer'sinstructions. Approximately 1 μg of gel purified Pfu FEN-1 PCR productwas digested with NcoI and PstI. After digestion, the DNA was purifiedusing the Geneclean II kit according to manufacturer's instructions. Onemicrogram of the pTrc99a vector was digested with NcoI and PstI prior toligation with the digested PCR product. One hundred nanograms ofdigested pTrc99a and 250 ng of digested Pfu FEN-1 PCR product werecombined and ligated to create pTrc99-PFFEN1. pTrc99-PFFEN1 was used totransform competent E. coli JM109 cells (Promega) using standardtechniques.

c) Cloning and Expression of a FEN-1 Endonuclease From Pyrococcus woesei

For the cloning of DNA encoding the Pyrococcus woesei (Pwo) FEN-1endonuclease, DNA was prepared from lyophilized P. woesei bacteria (DSMZ# 3773) as described (Zwickl et al., J. Bact., 172:4329 [1990]) withseveral changes. Briefly, one vial of P. woesei bacteria was rehydratedand resuspended in 0.5 ml of LB (Luria broth). The cells werecentrifuged at 14,000× g for 1 min and the cell pellet was resuspendedin 0.45 ml of TE. Fifty microliters of 10% SDS was added and the mixturewas incubated at RT for 5 min. The cell lysate was then extracted threetime with 1:1 phenol:chloroform and three times with chloroform. Fivehundred microliters of isopropanol was added to the extracted lysate andthe DNA was pelleted by centrifugation at 14,000× g for 10 min. The DNApellet was washed in 0.5 ml of 70% ethanol and the DNA was pelletedagain by centrifugation at 14,000× g for 5 min. The DNA pellet was driedand resuspended in 100 μl of TE and used for PCR reactions withoutfurther purification.

To generate a P. woesei FEN-1 gene fragment for cloning into anexpression vector, low stringency PCR was attempted with primerscomplementary to the ends of the P. furiosus FEN-1 gene open readingframe. The sequences of the 5′-end and 3′-end primers are5′-GATACCATGGGTGTCCCAATTGGTG-3′ (SEQ ID NO:80) and5′-TCGACGTCGACTTATCTCTTGAACCAACTTTCAAGGG-3′ (SEQ ID NO:81),respectively. The high level of sequence similarity of protein homologs(i.e., proteins other than FEN-1 proteins) from P. furiosus and P.woesei suggested that there was a high probability that the P. woeseiFEN-1 gene could be amplified using primers containing sequencescomplementary to the P. furiosus FEN-1 gene. However, this approach wasunsuccessful under several different PCR conditions.

The DNA sequence of FEN-1 genes from P. furiosus and M. jannaschii werealigned and blocks of sequence identity between the two genes wereidentified. These blocks were used to design internal primers (i.e.,complementary to sequences located internal to the 5′ and 3′ ends of theORF) for the FEN-1 gene that are complementary to the P. furiosus FEN-1gene in those conserved regions. The sequences of the 5′- and3′-internal primers are 5′-AGCGAGGGAGAGGCCCAAGC-3′ (SEQ ID NO:82) and5′-GCCTATGCCCTTTATTCCTCC-3′ (SEQ ID NO:83), respectively. A PCRemploying these internal primers was conducted using the Advantage™ PCRkit and resulted in production of a major band of ˜300 bp.

Since the PCR with the internal primers was successful, reactions wereattempted that contained mixtures of the internal (SEQ ID NOS:82 and 83)and external (SEQ ID NOS:80 and 81) primers. A reaction containing the5′-end external primer (SEQ ID NO:80) and 3′-end internal primer (SEQ IDNO:83) resulted in the production of a 600 bp band and a reactioncontaining the 5′-end internal primer (SEQ ID NO:82) and 3′-end externalprimer (SEQ ID NO:81) resulted in the production of a 750 bp band. Theseoverlapping DNA fragments were gel-purified and combined with theexternal primers (SEQ ID NOS:80 and 81) in a PCR reaction. This reactiongenerated a 1 kb DNA fragment containing the entire Pwo FEN-1 gene openreading frame. The resulting PCR product was gel-purified, digested, andligated exactly as described above for the Mja FEN-1 gene PCR product.The resulting plasmid was termed pTrc99-PWFEN1. pTrc99-PWFEN1 was usedto transform competent E. coli JM109 cells (Promega) using standardtechniques.

d) Cloning and Expression of a FEN-1 Endonuclease from Archaeoglobusfulgidus

The preliminary Archaeoglobus fulgidus (Afu) chromosome sequence of 2.2million bases was downloaded from the TIGR (The Institute for GenomicResearch) world wide web site, and imported into a software program(MacDNAsis), used to analyze and manipulate DNA and protein sequences.The unannotated sequence was translated into all 6 of the possiblereading frames, each comprising approximately 726,000 amino acids. Eachframe was searched individually for the presence of the amino acidsequence “VFDG” (valine, phenylalanine, aspartic acid, glycine), asequence that is conserved in the FEN-1 family. The amino acid sequencewas found in an open reading frame that contained other amino acidsequences conserved in the FEN-1 genes and that was approximately thesame size as the other FEN-1 genes. The ORF DNA sequence is shown in SEQID NO: 164, while the ORF protein sequence is shown in SEQ ID NO: 165.Based on the position of this amino acid sequence within the readingframe, the DNA sequence encoding a putative FEN-1 gene was identified.

The sequence information was used to design oligonucleotide primers thatwere used for PCR amplification of the FEN-1 -like sequence from A.fulgidus genomic DNA. Genomic DNA was prepared from A. fulgidus asdescribed in Ex. 29a for M. janaschii, except that one vial(approximately 5 ml of culture) of live A. fulgidus bacteria from DSMZ(DSMZ #4304) was used. One microliter of the genomic DNA was used forPCR reaction as described in Ex. 29a. The 5′ end primer is complementaryto the 5′ end of the Afu FEN-1 gene except it has a 1 base pairsubstitution to create an Nco I site. The 3′ end primer is complentaryto the 3′ end of the Afu FEN-1 gene downstream from the FEN-1 ORF exceptit contains a 2 base substitution to create a Sal I site. The sequencesof the 5′and 3′ end primers are 5′-CCGTCAACATTTACCATGGGTGCGGA-3′ (SEQ IDNO:166) and 5′-CCGCCACCTCGTAGTCGACATCCTTTTCGTG (SEQ ID NO:167),respectively.

Cloning of the resulting fragment was as described for the PfuFEN1 gene,above, to create the plasmid pTrc99-AFFEN1. The pTrcAfuHis plasmid wasconstructed by modifying pTrc99-AFFEN1, by adding a histidine tail tofacilitate purification. To add this histidine tail, standard PCRprimer-directed mutagenesis methods were used to insert the codingsequence for six histidine residues between the last amino acid codon ofthe pTrc99-AFFEN1 coding region and the stop codon. The resultingplasmid was termed pTrcAfuHis. The protein was then expressed asdescribed in Example 28f), and purified by binding to a Ni++ affinitycolumn, as described in Example 8.

e) Cloning and Expression of a FEN-1 Endonuclease from Methanobacteriumthermoautotrophicum

A tentative listing of all open reading frames of the Methanobacteriumthermoautotrophicum (Mth) genome on the Genome Therapeutics world wideweb page was searched for amino acid sequences conserved in the FEN-1genes. The amino acid sequence “VFDG” (valine, phenylalanine, asparticacid, glycine) was found in an open reading frame that also containedother conserved FEN-1 sequences. SEQ ID NO:260 provides the Mth FEN-1ORF DNA sequence as indicated by Genome Therapeutics, while SEQ IDNO:261 provides the Mth FEN-1 ORF protein sequence as indicated byGenome Therapeutics. However, this open reading frame was 259 aminoacids in length, as compared to the other archael FEN-1 genes, which areapproximately 325 amino acids long. To determine the cause of thisdiscrepancy, the DNA sequence for Mth FEN-1 was obtained in an identicalmanner as described above for Afu FEN-1.

Upon examination of the sequence, it was apparent that the open readingframe could be extended to 328 amino acids by deletion of a single baseat about position 750 of the open reading frame. The additional aminosequence added by deleting one base is 39% identical to the same regionof the P. furiosus FEN-1 gene. The DNA sequence of the putative MthFEN-1 gene was used to design oligonucleotide primers complementary tothe 5′ and 3′ ends of the gene. The 5′ oligonucleotide is complementaryto the 5′ end of the Mth FEN-1 gene except that it contains 2substitutions which create an NcoI site. The 3′ oligonucleotide iscomplementary to the 3′ end of the gene about 100 base pairs downstreamof where it is believed that the true open reading frame ends. Thisregion contains a natural PstI site. The sequences of the 5′ and 3′oligonucleotides are 5′-GGGTGTTCCCATGGGAGTTAAACTCAGG-3′ (SEQ ID NO:262)and 5′-CTGAATTCTGCAGAAAAAGGGG-3′ (SEQ ID NO:263), respectively.

Genomic DNA was prepared from 1 vial of frozen M. thermoautotrophicumbacteria from ATCC (ATCC # 29096) as described in Ex. 28a. PCR, cloning,expression, and purification of Mth FEN-1 was done as described inExamples 28a and 28f, except PstI was used instead of SalI. Theresulting plasmid was termed pTrc99-MTFEN1. Sequencing of the cloned MthFEN-1 gene revealed the presence of additional “T” nucleotide whencompared to the genome sequence published on the world wide web. This“T” residue at position 775 of the FEN-1 open reading frame causes aframe shift, creating the larger open reading frame that originallythought, based on comparison to the FEN genes from other organisms. SEQID NO:264 provides the sequence of the Mth ORF DNA sequence of thepresent invention, while SEQ ID NO:265 provides the sequence of the MthFEN-1 protein sequence of the present invention.

f) Large Scale Preparation of Recombinant Thermostable FEN-1 Proteins

The Mja, Pwo and Pfu FEN-1 proteins were purified by the followingtechnique, which is derived from a Taq DNA polymerase preparationprotocol (Engelke et al., Anal. Biochem., 191:396 [1990]) as follows. E.coli cells (strain JM109) containing either pTrc99-PFFEN1,pTrc99-PWFEN1, or pTrc99-MJFEN1 were inoculated into 3 ml of LB (LuriaBroth) containing 100 μg/ml ampicillin and grown for 16 hrs at 37° C.The entire overnight culture was inoculated into 200 ml or 350 ml of LBcontaining 100 μg/ml ampicillin and grown at 37° C. with vigorousshaking to an A₆₀₀ of 0.8. IPTG (1 M stock solution) was added to afinal concentration of 1 mM and growth was continued for 16 hrs at 37°C.

The induced cells were pelleted and the cell pellet was weighed. Anequal volume of 2× DG buffer (100 mM Tris-HCl, pH 7.6, 0.1 mM EDTA) wasadded and the pellet was resuspended by agitation. Fifty mg/ml lysozyme(Sigma, St. Louis, Mo.) was added to 1 mg/ml final concentration and thecells were incubated at room temperature for 15 min. Deoxycholic acid(10% solution) was added dropwise to a final concentration of 0.2% whilevortexing. One volume of H₂O and 1 volume of 2× DG buffer was added andthe resulting mixture was sonicated for 2 minutes on ice to reduce theviscosity of the mixture. After sonication, 3 M (NH₄)₂SO₄ was added to afinal concentration of 0.2 M and the lysate was centrifuged at 14000× gfor 20 min at 4° C. The supernatant was removed and incubated at 70° C.for 60 min at which time 10% polyethylimine (PEI) was added to 0.25%.After incubation on ice for 30 min., the mixture was centrifuged at14,000× g for 20 min at 4° C. At this point, the supernatant was removedand the FEN-1 proteins was precipitated by the addition of (NH₄)₂SO₄ asfollows.

For the Pwo and the Pfu FEN-1 preparations, the FEN-1 protein wasprecipitated by the addition of 2 volumes of 3 M (NH₄)₂SO₄. The mixturewas incubated overnight at room temperature for 16 hrs and the proteinwas centrifuged at 14,000× g for 20 min at 4° C. The protein pellet wasresuspended in 0.5 ml of Q buffer (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA,0.1% Tween 20). For the Mja FEN-1 preparation, solid (NH₄)₂SO₄ was addedto a final concentration of 3 M (˜75% saturated), the mixture wasincubated on ice for 30 min, and the protein was spun down andresuspended as described above.

The resuspended protein preparations were quantitated by determinationof the A₂₇₉ and aliquots containing 2-4 μg of total protein wereelectrophoresed on a 10% SDS polyacrylamide gel (29:1 acrylamide:bis-acrylamide) in standard Laemmli buffer [Laemmli, Nature 277:680[1970]) and stained with Coomassie Brilliant Blue R; the results areshown in FIG. 64.

In FIG. 64, lane 1 contains molecular weight markers (Mid-Range ProteinMolecular Weight Markers; Promega); the size of the marker proteins isindicated to the left of the gel. Lane 2 contains purified CLEAVASE BNnuclease; lanes 3-5 contain extracts prepared from E. coli expressingthe Pfu, Pwo and Mja FEN-1 nucleases, respectively. The calculated(i.e., using a translation of the DNA sequence encoding the nuclease)molecular weight of the Pfu FEN-1 nuclease is 38,714 daltons and thecalculated molecular weight for the Mja FEN-1 nuclease is 37,503Daltons. The Pwo and Pfu FEN-1 proteins co-migrated on the SDS-PAGE geland therefore, the molecular weight of the Pwo FEN-1 nuclease wasestimated to be 38.7 kDa.

g) Activity Assays Using FEN-1 Endonucleases

i) Mixed Hairpin Assay

The CLEAVASE BN nuclease has an approximately 60-fold greater affinityfor a 12 base pair stem-loop structure than an 8 base pair stem-loop DNAstructure. As a test for activity differences between the CLEAVASE BNnuclease and the FEN-1 nucleases, a mixture of oligonucleotides havingeither a 8 or a 12 bp stem-loop (see FIG. 60, which depicts the S-33 and11-8-0 oligonucleotides) was incubated with an extract prepared from E.coli cells overexpressing the Mja FEN-1 nuclease (prepared as describedabove). Reactions contained 0.05 μM of oligonucleotides S-33 (SEQ IDNO:84) and 11-8-0 (SEQ ID NO:85) (both oligonucleotides contained5′-fluorescein labels), 10 mM MOPS, pH 7.5, 0.05% Tween-20, 0.05% NP-40,I mM MnCl₂. Reactions were heated to 90° C. for 10 seconds, cooled to55° C., then 1 μl of crude extract (Mja FEN-1) or purified enzyme(CLEAVASE BN nuclease) was added and the mixtures were incubated at 55°C. for 10 minutes; a no enzyme control was also run. The reactions werestopped by the addition of formamide/EDTA, the samples wereelectrophoresed on a denaturing 20% acrylamide gel and visualized on aHitachi FMBIO 100 fluoroimager. The resulting image is shown in FIG. 65.

In FIG. 65, lane 1 contains the reaction products generated by theCLEAVASE BN nuclease, lane 2 contains the reaction products from the noenzyme control reaction and lane 3 contains the reaction productsgenerated by the Mja FEN-1 nuclease. The data shown in FIG. 76demonstrates that the CLEAVASE BN nuclease strongly prefers the S33structure (12 bp stem-loop) while the Mja FEN-1 nuclease cleavesstructures having either an 8 or a 12 bp stem-loop with approximatelythe same efficiency. This shows that the Mja FEN-1 nuclease has adifferent substrate specificity than the CLEAVASE BN nuclease, a usefulfeature for INVADER assays or CFLP®) analysis as discussed in theDescription of the Invention.

Example 29 Terminal Deoxynucleotidyl Transferase Selectively Extends theProducts of INVADER-Directed Cleavage

The majority of thermal degradation products of DNA probes will have aphosphate at the 3′-end. To investigate if the template-independent DNApolymerase, terminal deoxynucleotide transferase (TdT) can tail orpolymerize the aforementioned 3′-end phosphates (i.e., add nucleotidetriphosphates to the 3′ end) the following experiment was performed.

To create a sample containing a large percentage of thermal degradationproducts, the 5′ fluorescein-labeled oligonucleotide 34-078-01 (SEQ IDNO:86) (200 pmole) was incubated in 100 μl 10 mM NaCO₃ (pH 10.6), 50 mMNaCl at 95° C. for 13 hours. To prevent evaporation, the reactionmixture was overlaid with 60 μl ChillOut™ 14 liquid wax. The reactionmixture was then divided into two equal aliquots (A and B). Aliquot Awas mixed with one-tenth volume 3M NaOAc followed by three volumesethanol and stored at -20° C. Aliquot B was dephosphorylated by theaddition of 0.5 μl of 1M MgCl₂ and 1 μl of 1 unit/μl Calf IntestineAlkaline Phosphatase (CIAP) (Promega), with incubation at 37° C. for 30minutes. An equal volume of phenol:chloroform:isomayl alcohol (24:24: 1)was added to the sample followed by vortexing for one minute and thencentrifugation 5 minutes at maximum speed in a microcentrifuge toseparate the phases. The aqueous phase was removed to a new tube towhich one-tenth volume 3M NaOAc, and three volumes ethanol was addedfollowed by storage at −20° C. for 30 minutes. Both aliquots (A and B)were then centrifuged for 10 minutes at maximum speed in amicrocentrifuge to pellet the DNA. The pellets were then washed twotimes each with 80% ethanol and then desiccated to dryness. The driedpellets were then dissolved in 70 μl ddH₂O each.

The TdT reactions were conducted as follows. Six mixes were assembled,all mixes contained 10 mM TrisOAc (pH 7.5), 10 mM MgOAc, 50 mM KCl, and2 mM dATP. Mixes 1 and 2 contained one pmole of untreated 34-078-01 (SEQID NO:86), mixes 3 and 4 contained 2 μl of aliquot A (above), mixes 5and 6 contained 2 μl of aliquot B (above). To each 9 μl of mixes 1,3 and5, 1 μl ddH₂0 was added, to each 9 μl of mixes 2, 4, and 6, 1 μl of 20units/μl TdT (Promega) was added. The mixes were incubated at 37° C. for1 hour and then the reaction was terminated by the addition of 5 μl 95%formamide with 10 mM EDTA and 0.05% marker dyes. Five microliters ofeach mixture was resolved by electrophoresis through a 20% denaturingacrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer containing45 mM Tris-Borate (pH 8.3), 1.4 mM EDTA, and imaged using with the FMBIOImage Analyzer with a 505 nm filter. The resulting imager scan is shownin FIG. 66.

In FIG. 66, lanes 1, 3 and 5 contain untreated 34-078-01 (SEQ ID NO:86),heat-degraded 34-078-01, and heat-degraded, dephosphorylated, 34-078-01,respectively incubated in the absence of TdT. Lanes 2, 4 and 6 contain,untreated 34-078-01, heat-degraded 34-078-01, and heat-degraded,dephosphorylated, 34-078-01, respectively incubated in the presence ofTdT.

As shown in FIG. 66, lane 4, TdT was unable to extend thermaldegradation products that contain a 3′-end phosphate group, andselectively extends molecules that have a 3′-end hydroxyl group.

Example 30 Specific TdT Tailing of the Products of INVADER-DirectedCleavage with Subsequent Capture and Detection on NitrocelluloseSupports

When TdT is used to extend the specific products of cleavage, one meansof detecting the tailed products is to selectively capture the extensionproducts on a solid support before visualization. This Exampledemonstrates that the cleavage products can be selectively tailed by theuse of TdT and deoxynucleotide triphosphates, and that the tailedproducts can be visualized by capture using a complementaryoligonucleotide bound to a nitrocellulose support.

To extend the cleavage product produced in an INVADER-directed cleavagereaction, the following experiment was performed. Three reactionmixtures were assembled, each in a buffer of 10 mM MES (pH 6.5),0.5%Tween-20, 0.5% NP-40. The first mixture contained 5 fmols of targetDNA-M13mp18, 10 pmols of probe oligo 32-161-2 (SEQ ID NO:87; this probeoligonucleotide contains 3′ ddC and a Cy3 amidite group near the 3′end), and 5 pmols of INVADER oligonucleotide 32 161-1 (SEQ ID NO:88;this oligo contains a 3′ ddC). The second mixture contained the probeand INVADER oligonucleotides without target DNA. The third mixture wasthe same as the first mixture, and contained the same probe sequence,but with a 5′ fluorescein label (oligo 32-161-4 [SEQ ID NO:89; thisoligo contains a 3′ ddC, 5′ fluorescein label, and a Cy3 dye group nearthe 3′ end]), so that the INVADER-directed cleavage products could bedetected before and after cleavage by fluorescence imaging. The probeonly control sample contained 10 pmols of oligo 32-161-2 (SEQ ID NO:87).Each 3 μl of enzyme mix contained 5 ng of CLEAVASE DN nuclease in 7.5 mMMgCl₂. The TdT mixture (per each 4 pi) contained: 10 U of TdT (Promega),1 mM CoCl₂, 50 mM KCl, and 100 μM of dTTP. The INVADER cleavage reactionmixtures described above were assembled in thin wall tubes, and thereactions were initiated by the addition of 3 μl of CLEAVASE DN enzymemix. The reactions were incubated at 65° C. for 20 min. After cooling to37° C., 4 μl of the TdT mix was added and the samples were incubated for4 min at 37° C., Biotin-16-dUTP was then added to 100 μM and the sampleswere incubated for 50 min at 37° C. The reactions were terminated by theaddition of 1 μl of 0.5 M EDTA.

To test the efficiency of tailing the products were run on an acrylamidegel. Four microliters of each reaction mixture was mixed with 2.6 μl of95% formamide, 10 mM EDTA and 0.05% methyl violet and heated to 90° C.for 1 min, and 3 μl were loaded on a 20% denaturing acrylamide gel (19:1cross-linked ) with 7 M urea, in buffer containing 45 mM Tris-Borate (pH8.3), 1.4 mM EDTA. A marker (Φ×174-HinfI [fluorescein labeled]) also wasloaded. After electrophoresis, the gel was analyzed using a FMBIO-100Image Analyzer (Hitachi) equipped with a 505 nm filter. The resultingscan is shown in FIG. 67.

In FIG. 67, lane 1 contained the probe 32-161-2 only, without anytreatment. Lanes 2 and 3 contained the products of reactions run withouttarget DNA, without or with subsequent TdT tailing, respectively. Lanes4 and 5 contained the products of reactions run with target DNA, probeoligo 32-161-2 (SEQ ID NO:87) and INVADER oligo 32-161-1 (SEQ ID NO:88),without or with subsequent TdT tailing, respectively. Lanes 6 and 7 showthe products of reactions containing target DNA, probe oligo 32-161-4(SEQ ID NO:89) and INVADER oligo 32-161-1 (SEQ ID NO:88), without orwith subsequent TdT tailing, respectively. Lane M contains the markerΦ×174-HinfI.

The reaction products in lanes 4 and 5 are the same as those seen inlanes 6 and 7, except that the absence of a 5′ fluorescein on the probeprevents detection of the relased 5′ product (indicated as “A” near thebottom of the gel) or the TdT extended 5′ product (indicated as “B”,near the top of the gel). The Cy3-labeled 3′ portion of the cleavedprobe is visible in all of these reactions (indicated as “C”, just belowthe center of the gel).

To demonstrate detection of target-dependent INVADER-directed cleavageproducts on a solid support, the reactions from lanes 3 and 5 weretested on the Universal GENECOMB (Bio-Rad), which is a standardnitrocellulose matrix on a rigid nylon backing styled in a comb format,as depicted in FIG. 68. Following the manufacturer's protocol, with onemodification: 10 μl of the INVADER-directed cleavage reactions were usedinstead the recommended 10% of a PCR. To capture the cleavage products,2.5 pmols of the capture oligo 59-28-1 (SEQ ID NO:90) were spotted oneach tooth. The capture and visualization steps were conducted accordingto the manufacturer's directions. The results are shown in FIG. 68.

In FIG. 68, teeth numbered 6 and 7 show the capture results of reactionsperformed without and with target DNA present. Tooth 8 shows the kitpositive control.

The darkness of the spot seen on tooth 7, when compared to tooth 6,clearly indicates that products of INVADER-directed cleavage assays maybe specifically detected on solid supports. While the Universal GENECOMBwas used to demonstrate solid support capture in this instance, othersupport capture methods known to those skilled in the art would beequally suitable. For example, beads or the surfaces of reaction vesselsmay easily be coated with capture oligonucleotides so that they can thenbe used in this step. Alternatively, similar solid supports may easilybe coated with streptavidin or antibodies for the capture of biotin- orhapten-tagged products of the cleavage/tailing reaction. In any of theseembodiments, the products may be appropriately visualized by detectingthe resulting fluorescence, chemiluminescence, colorimetric changes,radioactive emissions, optical density change or any otherdistinguishable feature of the product.

Example 31 Comparison of the Effects of Invasion Length and 5′ Label ofthe Probe on INVADER-Directed Cleavage by the CLEAVASE A/G and Pfu FEN-1Nucleases

To investigate the effect of the length of invasion as well as theeffect of the type of dye on ability of Pfu FEN-1 and the CLEAVASE A/Gnuclease to cleave 5′ arms, the following experiment was performed.Three probes of similar sequences labeled with either fluorescein, TET,or Cy3, were assembled in reactions with three INVADER oligonucleotidesthat created overlapping target hybridization regions of eight, five,and three bases along the target nucleic acid, M13mp18.

The reactions were conducted as follows. All conditions were performedin duplicate. Enzyme mixes for Pfu FEN-1 and the CLEAVASE A/G nucleasewere assembled. Each 2 μl of the Pfu FEN-1 mix contained 100 ng of PfuFEN-1 (prepared as described in Ex. 28) and 7.5 mM MgCl₂. Each 2 μl ofthe CLEAVASE A/G mix contained 5.3 ng of the CLEAVASE A/G nuclease and4.0 mM MnCl₂. Six master mixes containing buffer, M13mp18, and INVADERoligonucleotides were assembled. Each 7 μl of mixes 1-3 contained 1 fmolM13mp18, 10 pmoles INVADER oligonucleotide (34-078-4 [SEQ ID NO:39],24-181-2 [SEQ ID NO:91], or 24-181-1 [SEQ ID NO:92], in 10 mM MOPS (pH7.5), 150 mM LiCl. Each 7 μl of mixes 4-6 contained 1 fmol of M13mp18,10 pmoles of INVADER oligonucleotide [34-078-4 (SEQ ID NO:39), 24-181-2(SEQ ID NO:91), or 24-181-1 (SEQ ID NO:92)] in 10 mM Tris (pH 8.0).Mixtures 1-6 were then divided into three mixtures each, to which wasadded either the fluorescein-labeled probe (oligo 34-078-01; SEQ IDNO:86), the Cy3-labeled probe (oligo 43-20; SEQ ID NO:93) or theTET-labeled probe (oligo 90; SEQ ID NO:32 containing a 5′ TET label).Each 7 μl of all mixtures contained 10 pmoles of corresponding probe.The DNA solutions described above were covered with 10 μl of CHILLOUTevaporation barrier and brought to 68° C.

The reactions made from mixes 1-3 were started with 2 μl of the CLEAVASEA/G nuclease mix, and the reactions made from mixes 4-6 were startedwith 2 μl of the Pfu FEN-1 mix. After 30 minutes at 68° C., thereactions were terminated by the addition of 8 μl of 95% formamide with10 mM EDTA and 0.05% marker dyes. Samples were heated to 90° C. for 1minute immediately before electrophoresis through a 20% denaturingacrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer containing45 mM Tris-Borate (pH 8.3), 1.4 mM EDTA. The products of the cleavagereactions were visualized following electrophoresis by the use of aHitachi FMBIO fluorescence imager. Results from the fluorescein-labeledprobe are shown in FIG. 69, results from the Cy3-labeled probe in FIG.70, and results from the TET-labeled probe in FIG. 71. In each of theseFigures, the products of cleavage by CLEAVASE A/G are shown in lanes 1-6and the products of cleavage by PfuFEN-1 are shown in lanes 7-12. Ineach in case the uncut material appears as a very dark band near the topof the gel, indicated by a “U” on the left. The products of cleavagedirected by INVADER oligonucleotides with 8, 5 or 3 bases of overlap(i.e., the “X” region was 8, 5, or 3 nt long) are shown in the first,second and third pair of lanes in each set, respectively and thereleased labeled 5′ ends from these reactions are indicated by thenumbers 8, 5, and 3 on the left. Note that in the cleavage reactionsshown in FIG. 70 the presence of the positively charged Cy3 dye causesthe shorter products to migrate more slowly than the larger products.These products do not contain any additional positive charges (e.g.,amino modifications as used in Example 23), and thus still carry a netnegative charge, and migrate towards the positive electrode in astandard electrophoresis run.

It can be seen from these data that the CLEAVASE A/G and Pfu FEN-1structure-specific nucleases respond differently to both dye identityand to the size of the piece to be cleaved from the probe. The Pfu FEN-1nuclease showed much less variability in response to dye identity thandid the CLEAVASE A/G nuclease, showing that any dye wold be suitable foruse with this enzyme. In contrast, the amount of cleavage catalyzed bythe CLEAVASE A/G nuclease varied substantially with dye identity. Use ofthe fluorescein dye gave results very close to those seen with the PfuFEN-1 nuclease, while the use of either Cy3 or TET gave dramaticallyreduced signal when compared to the Pfu FEN-1 reactions. The oneexception to this was in the cleavage of the 3 nt product carrying a TETdye (lanes 5 and 6, FIG. 71), in which the CLEAVASE A/G nuclease gavecleavage at the same rate as the Pfu FEN-1 nuclease. These data indicatethat, while CLEAVASE A/G may be used to cleave probes labeled with theseother dyes, the Pfu FEN-1 nuclease is a preferred nuclease for cleavageof Cy3- and TET-labeled probes.

Example 32 Examination of the Effects of a 5′ Positive Charge on theRate of Invasive Cleavage Using the CLEAVASE A/G or Pfu FEN-1 Nucleases

To investigate whether the positive charges on 5′ end of probeoligonucleotides containing a positively charged adduct(s) (i.e., chargereversal technology or CRT probes as described in Ex. 23 and 24 have aneffect on the ability of the CLEAVASE A/G or Pfu FEN-1 nucleases tocleave the 5′ arm of the probe, the following experiment was performed.

Two probe oligonucleotides having the following sequences were utilizedin INVADER reactions: Probe 34-180-1:(N-Cy3)T_(NH2)T_(NH2)CCAGAGCCTAATTTGCC AGT(N-fluorescein)A, where Nrepresents a spacer containing either the Cy3 or fluorescein group (SEQID NO:94) and Probe 34-180-2: 5′-(N-TET)TTCCAGAGCCTAATTTGCCAGT-(N-fluorescein)A, where N represents a spacer containingeither the TET or fluorescein group (SEQ ID NO:95). Probe 34-180-1 hasamino-modifiers on the two 5′ end T residues and a Cy3 label on the 5′end, creating extra positive charges on the 5′ end. Probe 34-180-2 has aTET label on the 5′ end, with no extra positive charges. The fluoresceinlabel on the 3′ end of probe 34-180-1 enables the visualization of the3′cleaved products and uncleaved probes together on an acrylamide gelrun in the standard direction (i.e., with the DNA migrating toward thepositive electrode). The 5′ cleaved product of probe 34-180-1 has a netpositive charge and will not migrate in the same direction as theuncleaved probe, and is thus visualized by resolution on a gel run inthe opposite direction (i.e.; with this DNA migrating toward thenegative electrode).

The cleavage reactions were conducted as follows. All conditions wereperformed in duplicate. Enzyme mixes for the Pfu FEN-1 and CLEAVASE A/Gnucleases were assembled. Each 2 μl of the Pfu FEN-1 mix contained 100ng of Pfu FEN-1 (prepared as described in Ex. 28) and 7.5 mM MgCl₂. Each2 μl of the CLEAVASE A/G nuclease mix contained 26.5 ng of CLEAVASE A/Gnuclease and 4.0 mM MnCl₂. Four master mixes containing buffer, M13mp18,and INVADER oligonucleotides were assembled. Each 7 μl of mix 1contained 5 fmol M13mp18, 10 pmoles INVADER oligonucleotide 123 (SEQ IDNO:96) in 10 mM HEPES (pH 7.2). Each 7 μl of mix 2 contained 1 fmolM13mp18, 10 pmoles INVADER oligonucleotide 123 in 10 mM HEPES (pH 7.2).Each 7 μl of mix 3 contained 5 fmol M13mp18, 10 pmoles INVADERoligonucleotide 123 in 10 mM HEPES (pH 7.2), 250 mM KGlu. Each 7 μl ofmix 4 contained 1 fmol M13mp18, 10 pmoles INVADER oligonucleotide 123 in10 mM HEPES (pH 7.2), 250 mM KGlu. For every 7 μl of each mix, 10 pmolesof either probe 34-180-1 (SEQ ID NO:94) or probe 34-180-2 (SEQ IDNO:95)was added. The DNA solutions described above were covered with 10 μl ofCHILLOUT evaporation barrier and brought to 65° C. The reactions madefrom mixes 1-2 were started by the addition of 2 μl of the Pfu FEN-1mix, and the reactions made from mixes 3-4 were started by the additionof 2 μl of the CLEAVASE A/G nuclease mix. After 30 minutes at 65° C.,the reactions were terminated by the addition of 8 μl of 95% formamidecontaining 10 mM EDTA. Samples were heated to 90° C. for 1 minuteimmediately before electrophoresis through a 20% denaturing acrylamidegel (19:1 cross-linked) with 7 M urea, in a buffer containing 45 mMTris-Borate (pH 8.3), 1.4 mM EDTA and a 20% native acrylamide gel (29:1cross-linked) in a buffer containing 45 mM Tris-Borate (pH 8.3), 1.4 mMEDTA.

The products of the cleavage reactions were visualized followingelectrophoresis by the use of a Hitachi FMBIO fluorescence imager. Theresulting images are shown in FIG. 72. FIG. 72A shows the denaturinggel, which was run in the standard electrophoresis direction, and FIG.72B shows the native gel, which was run in the reverse direction. Thereaction products produced by Pfu FEN-1 and CLEAVASE A/G nucleases areshown in lanes 1-8 and 9-16, respectively. The products from the 5 fmolM13mp18 and 1 fmol M13mp18 reactions are shown in lanes 1-4, 9-12 (5fmol) and 5-8, 13-16 (1 fmol). Probe 34-180-1 is in lanes 1-2, 5-6,9-10, 13-14 and probe 34-180-2 is in lanes 3-4,7-8, 11-12, 15-16.

The fluorescein-labeled 3′ end fragments from all cleavage reactions areshown in FIG. 72A, indicated by a “3′” mark at the left. The 3 nt 5′TET-labeled products are not visible in this Figure, while the 5′Cy3-labeled products are shown in FIG. 72B.

The 3′ end bands in FIG. 72A can be used to compare the rates ofcleavage by the different enzymes in the presence of the different 5′end labels. It can be seen from this band that regardless of the amountof target nucleic acid present, both the Pfu FEN-1 and the CLEAVASE A/Gnucleases show more product from the 5′ TET-labeled probe. With the PfuFEN-1 nuclease this preference is modest, with only an approximately 25to 40% increase in signal. In the case of the CLEAVASE A/G nuclease,however, there is a strong preference for the 5′ TET label. Therefore,although when the charge reversal method is used to resolve theproducts, a substantial amount of product is observed from the CLEAVASEA/G nuclease-catalyzed reactions, the Pfu FEN-1 nuclease is a preferredenzyme for cleavage of Cy3-labeled probes.

Example 33 The Use of Universal Bases in the Detection of Mismatches byINVADER Directed Cleavage

The term “degenerate base” refers to a base on a nucleotide that doesnot hydrogen bond in a standard “Watson-Crick” fashion to a specificbase complement (i.e., A to T and G to C). For example, the inosine basecan be made to pair via one or two hydrogen bonds to all of the naturalbases (the “wobble” effect) and thus is called degenerate.Alternatively, a degenerate base may not pair at all; this type of basehas been referred to as a “universal” base because it can be placedopposite any nucleotide in a duplex and, while it cannot contributestability by base-pairing, it does not actively destabilize by crowdingthe opposite base. Duplexes using these universal bases are stabilizedby stacking interactions only. Two examples of universal bases,3-nitropyrrole and 5-nitroindole, are shown in FIG. 73. Inhybridization, placement of a 3-nitropyrrole three bases from a mismatchposition enhances the differential recognition of one base mismatches.The enhanced discrimination seems to come from the destabilizing effectof the unnatural base (i.e., an altered T_(m) in close proximity to themismatch). To test this same principle as a way of sensitively detectingmismatches using the INVADER-directed cleavage assay, INVADERoligonucleotides were designed using the universal bases shown in FIG.73, in the presence or absence of a natural mismatch. In theseexperiments, the use of single nitropyrrole bases or pairs ofnitroindole bases that flank the site of the mismatch were examined.

The target, probe and INVADER oligonucleotides used in these assays areshown in FIG. 74. A 43 nucleotide oligonucleotide (oligo 109; SEQ IDNO:97) was used as the target. The probe oligonucleotide (oligo 61; SEQID NO:50) releases a net positively charged labeled product uponcleavage. In FIG. 74, the INVADER oligonucleotide is shown schematicallyabove the target oligonucleotide as an arrow; the large arrowheadindicates the location of the mismatch between the INVADER oligos andthe target. Under the target oligonucleotide, the completelycomplementary, all natural (i.e., no universal bases) INVADER oligo(oligo 67; SEQ ID NO:51) and a composite of INVADER oligos containinguniversal bases (“X”) on either side of the mismatch (“M”) are shown.The following INVADER oligos were employed: oligo 114 (SEQ ID NO:98),which contains a single nt mismatch; oligo 115 (SEQ ID NO:99), whichcontains two 5-nitroindole bases and no mismatch; oligo 116 (SEQ ID NO:100), which contains two 5-nitroindole bases and a single nt mismatch;oligo 112 (SEQ ID NO: 101), which contains one 3-nitropyrrole base andno mismatch; oligo 113 (SEQ ID NO: 102), which contains one5-nitropyrrole base and a single nt mismatch; and oligo 67 (SEQ IDNO:51), which is completely complementary to the target.

The INVADER-directed cleavage reactions were carried out in 10 μl of 10mM MOPS (pH 7.2), 100 mM KCl, containing 1 μM of the appropriateinvading oligonucleotide (oligos 67, 112-116), 10 nM synthetic target109, 1 μM Cy-3 labeled probe 61 and 2 units of CLEAVASE DV (prepared asdescribed in Ex. 27). The reactions were overlayed with Chill-Out®liquid wax, brought to the appropriate reaction temperature, 52° C., 55°C., or 58° C. and initiated with the addition of 1 μl of 40 mM MnCl₂.Reactions were allowed to proceed for 1 hour and were stopped by theaddition of 10 μl formamide. One fourth of the total volume of eachreaction was loaded onto 20% non-denaturing polyacrylamide gels, whichwere electrophoresed in the reverse direction. The products werevisualized using an Hitachi FMBIO-100 fluorescent scanner using a 585 nmfilter. The resulting images are shown in FIGS. 75A-C. In each panel,lanes 1-6 contain reactions products from reactions using INVADER oligo67, 114, 115, 116, 112 and 113, respectively. Reactions run at 52° C.,55° C. and 58° C. are shown in Panels A, B and C, respectively.

These data show that two flanking 5-nitroindoles display a significantlygreater differentiation then does the one 3-nitropyrrole system, or theall natural base hybridization, and this increased sensitivity is nottemperature dependent. This demonstrates that the use of universal basesis a useful means of sensitively detecting single base mismatchesbetween the target nucleic acid and the complex of detectionoligonucleotides of the present invention.

Example 34 Detection of Point Mutations in the Human Ras Oncogene Usinga Miniprobe

It is demonstrated herein that very short probes can be used forsensitive detection of target nucleic acid sequences (Ex. 37). In thisExample, it is demonstrated that the short probes work very poorly whenmismatched to the target, and thus can be used to distinguish a givennucleic acid sequence from a close relative with only a single basedifference. To test this system synthetic human ras oncogene targetsequences were created that varied from each other at one position.Oligonucleotide 166 (SEQ ID NO:103) provided the wild-type ras targetsequence. Oligonucleotide 165 (SEQ ID NO:104) provided the mutant rastarget sequence. The sequence of these oligonucleotides are shown inFIG. 76, and the site of the sequence variation in the sitecorresponding to codon 13 of the ras gene is indicated. The INVADERoligonucleotide (oligo 162) has the sequence:5′-G_(s)C_(s)T_(s)C_(s)A_(s)A_(s)G_(s)G_(s)C_(s)ACTCTTGCC TACGA-3′ (SEQID NO:105), where the “S” indicates thiol linkages (i.e., these are2′-deoxynucleotide-5′-O-(1-thiomonophates)). The miniprobe (oligo 161)has the sequence: 5′-(N-Cy3) T_(NH2)T_(NH2)CACCAG-3′ (SEQ ID NO:106) andis designed to detect the mutant ras target sequence (i.e., it iscompletely complementary to oligo 165). The stacker oligonucleotide(oligo 164) has the sequence:5′-C_(s)T_(s)C_(s)C_(s)A_(s)A_(s)C_(s)T_(s)A_(s)CCACAAGTTTATATTCAG-3′(SEQID NO:107). A schematic showing the assembly of these oligonucleotidesinto a cleavage structure is depicted in FIG. 76.

Each cleavage reaction contained 100 nM of both the invading (oligo 162)and stacking (oligo 164) oligonucleotides, 10 μM Cy3-labeled probe(oligo 161) and 100 pM of either oligo 165 or oligo 166 (target DNA) in10 μl of 10 mM HEPES (pH 7.2), 250 mM KGlu, 4 mM MnCl₂. The DNA mixtureswere overlaid with mineral oil, heated to 90° C. for 15 sec then broughtto a reaction temperature of 47°, 50°, 53° or 56° C. Reactions wereinitiated by the addition of 1 μl of 100 ng/μl Pfu FEN-1. Reactions wereallowed to proceed for 3 hours and stopped by the addition of 10 μlformamide. One fourth of the total volume od each reaction was loadedonto a 20% non-denaturing polyacrylamide gel, which was electrophoresedin the reverse direction. The gel was scanned using an Hitachi FMBIO-100fluorescent scanner fitted with a 585 nm filter, and the resulting imageis shown in FIG. 77.

In FIG. 77, for each reaction temperature tested, the products fromreactions containing either the mutant ras target sequence (oligo 165)or the wild-type (oligo 166) are shown.

These data demonstrate that the miniprobe can be used to sensitivelydiscriminate between sequences that differ by a single nucleotide. Theminiprobe was cleaved to produce a strong signal in the presence of themutant target sequence, but little or no miniprobe was cleaved in thepresence of the wild-type target sequence. Furthermore, thediscrimination between closely related targets is effective over atemperature range of at least 10° C., which is a much broader range oftemperature than can usually be tolerated when the selection is based onhybridization alone (e.g., hybridization with ASOs). This suggests thatthe enzyme may be a factor in the discrimination, with the perfectlymatched miniprobe being the preferred substrate when compared to themismatched miniprobe. Thus, this system provides sensitive and specificdetection of target nucleic acid sequences.

Example 35 Effects of 3′ End Identity on Site of Cleavage of a ModelOligonucleotide Structure

As described in the Examples above, structure-specific nucleases cleavenear the junction between single-stranded and base-paired regions in abifurcated duplex, usually about one base pair into the base-pairedregion. It was shown in Example 10 that thermostable 5′ nucleases,including those of the present invention (e.g., CLEAVASE BN nuclease,CLEAVASE A/G nuclease), have the ability to cleave a greater distanceinto the base paired region when provided with an upstreamoligonucleotide bearing a 3′region that is homologous to a 5′ region ofthe subject duplex, as shown in FIG. 26. It has also been determinedthat the 3′ terminal nucleotide of the INVADER oligonucleotide may beunpaired to the target nucleic acid, and still shift cleavage the samedistance into the down stream duplex as when paired. It is shown in thisExample that it is the base component of the nucleotide, not the sugaror phosphate, that is necessary to shift cleavage.

FIGS. 78A and B shows a synthetic oligonucleotide that was designed tofold upon itself, and that consists of the following sequence:5′-GTTCTCTGCTCTCTGGTC GCTGTCTCGCTTGTGAAACAAGCGAGACAGCGTGGTCTCTCG-3′ (SEQID NO:29). This oligonucleotide is referred to as the “S-60 Hairpin.”The 15 basepair hairpin formed by this oligonucleotide is furtherstabilized by a “tri-loop” sequence in the loop end (i.e., threenucleotides form the loop portion of the hairpin) (Hiraro et al.,Nucleic Acids Res., 22(4): 576 [1994]). FIG. 78B shows the sequence ofthe P-15 oligonucleotide (SEQ ID NO:30) and the location of the regionof complementarity shared by the P-15 and S-60 hairpin oligonucleotides.In addition to the P-15 oligonucleotide shown, cleavage was also testedin the presence of the P-14 oligonucleotide (SEQ ID NO:108) (P-14 is onebase shorter on the 3′ end as compared to P-15), the P-14 with an abasicsugar (P-14d; SEQ ID NO:109) and the P14 with an abasic sugar with a 3′phosphate (P-14 dp; SEQ ID NO:110). A P-15 oligo with a 3′ phosphate,P-15 p (SEQ ID NO:111) was also examined. The black arrows shown in FIG.78 indicate the sites of cleavage of the S-60 hairpin in the absence(top structure; A) or presence (bottom structure; B) of the P-15oligonucleotide.

The S-60 hairpin molecule was labeled on its 5′ end with fluorescein forsubsequent detection. The S-60 hairpin was incubated in the presence ofa thermostable 5′ nuclease in the presence or the absence of the P-15oligonucleotide. The presence of the full duplex that can be formed bythe S-60 hairpin is demonstrated by cleavage with the CLEAVASE BN 5′nuclease, in a primer-independent fashion (i.e., in the absence of theP-15 oligonucleotide). The release of 18 and 19-nucleotide fragmentsfrom the 5′ end of the S-60 hairpin molecule showed that the cleavageoccurred near the junction between the single and double strandedregions when nothing is hybridized to the 3′ arm of the S-60 hairpin(FIG. 27, lane 2).

The reactions shown in FIG. 78C were conducted in 10 μl 1X CFLP bufferwith 1 mM MnCl₂ and 50 mM K-Glutamate, in the presence of 0.02 μM S-60,0.5 μM INVADER oligonucleotide and 0.01 ng per μl CLEAVASE BN nuclease.Reactions were incubated at 40° C. for 5 minutes and stopped by theaddition of 8 μl of stop buffer (95% formamide, 20 mM EDTA, 0.02% methylviolet). Samples were heated to 75° C. for 2 min immediately beforeelectrophoresis through a 15% acrylamide gel (19:1 cross-linked), with 7M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Gels werethen analyzed with a FMBIO-100 Image Analyzer (Hitachi) equipped with505 nm filter. The resulting image is shown in FIG. 78C.

In FIG. 78C lane 1 contains products from the no enzyme control; lane 2contains products from a reaction run in the absence of an INVADERoligo; lanes 3-6 contain products from reactions run the presence of theP-14d, P-14dp, P-15 and P-15p INVADER oligos, respectively.

From the data shown in FIG. 78C, it can be seen that the use of the P-15INVADER oligonucleotide produces a shift in the cleavage site, while theP14 INVADER oligonucleotide with either a ribose (P14d) or aphosphorylated ribose (P14dp) did not This indicates that the 15thresidue of the INVADER oligonucleotide must have the base group attachedto promote the shift in cleavage. Interestingly, the addition ofphosphate to the 3′ end of the P15 oligonucleotide apparently reversedthe shifting of cleavage site. The cleavage in this lane may in fact becleavage in the absence of an INVADER oligonucleotide as is seen in lane2. In experiments with 5′ dye-labeled INVADER oligonucleotides with 3′phosphate groups these oligonucleotides have been severely retarded ingel migration, suggesting that either the enzyme or another constituentof the reaction (e.g., BSA) is able to bind the 3′ phosphateirrespective of the rest of the cleavage structure. If the INVADERoligonucleotides are indeed being sequestered away from the cleavagestructure, the resulting cleavage of the S-60 hairpin would occur in a“primer-independent” fashion, and would thus not be shifted.

In addition to the study cited above, the effects of other substituentson the 3′ ends of the INVADER oligonucleotides were investigated in thepresence of several different enzymes, and in the presence of eitherMn++ or Mg++. The effects of these 3′ end modifications on thegeneration of cleaved product are summarized in the following table. Allof modifications were made during standard oligonucleotide synthesis bythe use of controlled pore glass (CPG) synthesis columns with the listedchemical moiety provided on the support as the synthesis startingresidue. All of these CPG materials were obtained from Glen ResearchCorp. (Sterling, Va.).

FIG. 79 provides the structures for the 3′ end substituents used inthese experiments.

TABLE 4 Modification Studies At 3′ End of INVADER Oligo Effect onINVADER Rxn. (As Extension By INVADER) Enzyme:Condition- 3′-EndModifiaction Terminal Transferase Effect 3′ phosphate no A:5-inhibitsreaction, Glen part #20-2900-42 no detectable activity 3′ acridine yes,poorly A:5-decrease in activity, <10% Glen part #20-2973-42 B:5-decreasein activity, <10% B:4-decrease in activity, <10% C:1-decrease inactivity, <10% C:2-decrease in activity, ~20% C:4-decrease in activity,~50% C:3-decrease in activity, <5% 3′ carboxylate no A:1-decrease inactivity, ~50% Glen part #20-4090-42 activity shift in cleavage siteC:3-reduces rate, <10% activity 3′ nitropyrole yes A:5-increase inactivity, ~2X Glen part #20-2143-42 3′ nitroindole yes A:5-decrease inactivity, ~33% Glen part #20-2144-42 activity 3′ arabinose yesA:5-decrease in activity, ~50% Glen part #10-4010-90 activity3′dideoxyUTP- no A:5-decrease in activity, ~40% fluorescein activity3′-3′ linkage no A:1-equivalent cleavage Glen part #20-0002-01 activityshift in cleavage site C:3-decrease in activity, ~25% activity 3′glyceryl yes, very poorly C:3-decrease in activity, ~30% Glen part#20-2902-42 activity loss of specificity of cleav- age (2 sites) 3′amino modifier C7 yes C:3-decrease in activity, ~30% Glen part#20-2957-42 activity loss of specificity, multiple sites 3′ phosphate noA:5-inhibitis reaction, Glen part #20-2900-42 no detectable activity3′deoxy, 2′OH yes, very poorly A:5-decreases in activity, <20% Glen part#20-2104-42 activity B:5-decrease in activity, <20% activityB:3-decrease in activity, <20% activity C:1-equivalent activityC:2-equivalent activity C:4-increase in activity C:3-decrease inactivity, ~40% activity Enzymes: A) CLEAVASE DV nuclease B) CLEAVASE BNnuclease C) Pfu FEN-1 Condition: 1) 4 mM MnCl₂, 150 mM LiCl 2) 4 mMMnCl₂, 50 mM KCl 3) 7.5 mM MgCl₂, no monovalent 4) 4 mM MgCl₂, 50 mM KCl5) 10 mM MgOAc, 50 mM KCl

It can be seen from these data that many different modifications can beused on the 3′ end of the INVADER oligonucleotide without detriment. Invarious embodiments of the present invention, such 3′ end modificationsmay be used to block, facilitate, or otherwise alter the hybridizationcharacteristics of the INVADER oligonucleotide, (e.g., to increasediscrimination against mismatches, or to increase tolerance ofmismatches, or to tighten the association between the INVADERoligonucleotide and the target nucleic acid). Some substituents may beused to alter the behavior of the enzyme in recognizing and cleavingwithin the assembled complex.

Altered 3′ ends may also be used to prevent extension of the INVADERoligonucleotide by either template-dependent or template-independentnucleic acid polymerases. The use of otherwise unmodifieddideoxynucleotides (i.e., without attached dyes or other moieties) are aparticularly preferred means of blocking extension of INVADERoligonucleotides, because they do not decrease cleavage activity, andthey are absolutely unextendable.

Example 36 Effect of Probe Concentration, Temperature and a StackerOligonucleotide on the Cleavage of Miniprobes by INVADER-DirectedCleavage

The stacker oligonucleotides employed to form cleavage structures mayserve two purposes in the detection of a nucleic acid target using aminiprobe. The stacker oligonucleotide may help stabilize theinteraction of the miniprobe with the target nucleic acid, leading togreater accumulation of cleaved probe. In addition, the presence of thisoligo in the complex elongates the duplex downstream of the cleavagesite, which may enhance the cleavage activity of some of the enzymes ofthe present invention. An example of different preferences for thelength of this duplex by different structure-specific nucleases is seenin the comparison of the CLEAVASE BN nuclease and the Mja FEN-1 nucleasecleavage of 8 bp and 12 bp duplex regions in FIG. 65. Increased affinityof the enzyme for the cleavage structure also results in increasedaccumulation of cleaved probe during reactions done for a set amount oftime.

The amount of miniprobe binding to the target is also affected by theconcentration of the miniprobe in the reaction mixture. Even when aminiprobe is only marginally likely to hybridize (e.g., when thereaction is performed at temperatures in excess of the expected meltingtemperature of the probe/target duplex), the amount of probe on thetarget at any given time can be increased by using high concentrationsof the miniprobe.

The need for a stacker oligonucleotide to enhance cleavage of theminiprobe was examined at both low and high probe concentrations. Thereactions were carried out in 10 μl of 10 mM HEPES (pH 7.2), 250 mMKGIu, 4 mM MnCl₂, containing 100 nM of both the invading (oligo 135; SEQID NO:112) and stacking oligonucleotides (oligo 147; SEQ ID NO:113) and100 pM ssM13 DNA. The reactions were overlayed with mineral oil, heatedto 90° C. for 15 sec then brought to the reaction temperature. Reactionswere performed at 35°, 40°, 45°, 50°, 55°, 60°, and 65° C. The cleavagereactions were initiated by the addition of 1 μl of 100 ng/μl Pfu FEN-Iand 1 μl of varying concentrations of Cy-3 labeled 142 miniprobeoligonucleotide (SEQ ID NO:114). Reactions were allowed to proceed for 1hour and stopped by the addition of 10 μl formaldehyde. One fourth ofthe total volume of each reaction was loaded onto 20% non-denaturingpolyacrylamide gels, which were electrophoresed in the reversedirection. Gels were visualized using an Hitachi FMBIO-100 fluorescentscanner using a 585 nm filter. The fluorescence in each product band wasmeasured and the graph shown in FIG. 80 was created using a MicrosoftExcel spreadsheet.

The data summarized in FIG. 80 showed that the concentration of theminiprobe had a significant effect on the final measure of product,showing dramatic increases as the concentration was raised. Increases inthe concentration of the miniprobe also shifted the optimum reactiontemperature upward. It is known in the art that the concentration of thecomplementary strands in a hybridization will affect the apparent Tm ofthe duplex formed between them. More significantly to the methods andcompositions of the present invention is the fact that the presence ofthe stacker oligonucleotide has a profound influence on the cleavagerate of the miniprobe at all probe concentrations. At each of the probeconcentrations the presence of the stacker as much as doubled the signalfrom the cleavage product. This demonstrated the utility of using thestacker oligonucleotide in combination with the miniprobes describedherein.

Example 37 The Presence of a Mismatch in the INVADER OligonucleotideDecreases the Cleavage Activity of the CLEAVASE A/G Nuclease

In any nucleic acid detection assay it is of additional benefit if theassay can be made to sensitively detect minor differences betweenrelated nucleic acids. In the following experiment, model cleavagesubstrates were used that were identical except for the presence orabsence of a mismatch near the 3′ end of the INVADER oligonucleotidewhen hybridized to the model target nucleic acid. The effect of amismatch in this region on the accumulation of cleaved probe was thenassessed.

To demonstrate the effect of the presence of a mismatch in the INVADERoligonucleotide on the ability of the CLEAVASE A/G nuclease to cleavethe probe oligonucleotide in an INVADER assay the following experimentwas conducted. Cleavage of the test oligonucleotide IT-2 (SEQ ID NO:115)in the presence of INVADER oligonucleotides IT-1 (SEQ ID NO:116) andIT-1A4 (SEQ ID NO:117). Oligonucleotide IT-1 is fully complementary tothe 3′ arm of IT-2, whereas oligonucleotide IT-1A4 has a T->Asubstitution at position 4 from the 3′ end that results in an A/Amismatch in the INVADER-target duplex. Both the matched and mismatchedINVADER oligonucleotides would be expected to hybridize at thetemperature at which the following reaction was performed. FIG. 81provides a schematic showing IT-1 annealed to the folded IT-2 structureand showing IT-1A4 annealed to the folded IT-2 structure.

The reactions were conducted as follows. Test oligonucleotide IT-2 (0.1μM), labeled at the 5′ end with fluorescein (Integrated DNATechnologies), was incubated with 0.26 ng/μl CLEAVASE AG in 10 μl ofCFLP® buffer with 4 mM MgCl₂, in the presence of 1 μM IT-1 or IT-1A4 at40° C. for 10 min; a no enzyme control was also run. Samples wereoverlaid with 15 μl Chill-Out® liquid wax to prevent evaporation.Reactions were stopped by addition of 4 μl stop buffer (95% formamide,20 mM EDTA, 0.02% methyl violet). The cleavage products were separatedon a 20% denaturing polyacrylamide gel and analyzed with the FMBIO-100Image Analyzer (Hitachi) equipped with 505 nm filter. The resultingimage is shown in FIG. 82.

In FIG. 82, lane 1 contains reaction products from the no enzyme controland shows the migration of the uncut IT-2 oligo; lanes 2-4 containproducts from reactions containing no INVADER oligo, the IT-1 INVADERoligo and the IT-1A4 INVADER oligo, respectively.

These data show that cleavage is markedly reduced by the presence of themismatch, even under conditions in which the mismatch would not beexpected to disrupt hybridization. This demonstrates that the INVADERoligonucleotide binding region is one of the regions within the complexin which can be used for mismatch detection, as revealed by a drop inthe cleavage rate.

Example 38 Comparison of the Activity of the Pfu FEN-1 And Mja EN-1Nucleases in the INVADER Reaction

To compare the activity of the Pfu FEN-1 and the Mja FEN-1 nucleases inINVADER reaction the following experiment was performed. A testoligonucleotide IT3 (SEQ ID NO:118) that forms an INVADER-Target hairpinstructure and probe oligonucleotide PR1 (SEQ ID NO:119) labeled at the5′ end with fluorescein (Integrated DNA Technologies) were employed inINVADER assays using either the Pfu FEN-1 or the Mja FEN-1 nucleases.

The assays were conducted as follows. Pfu FEN-1 (13 ng/μl) and Mja FEN-1(10 ng/μl) (prepared as described in Ex. 28) were incubated with the IT3(0.1 nM) and PR1 (2 and 5 μM) oligonucleotides in 10 μL CFLP® buffer, 4mM MgCl₂, 20 mg/ml tRNA at 55° C. for 41 min. Samples were overlaid with15 μl Chill-Out® evaporation barrier to prevent evaporation. Reactionswere stopped by addition of 70 μl stop buffer (95% formamide, 20 mMEDTA, 0.02% methyl violet). Reaction products (1 μl) were separated on a20% denaturing polyacrylamide gel, visualized using a fluoroimager andthe bands corresponding to the probe and the product were quantitiated.The resulting image is shown in FIG. 83. In FIG. 83, the turnover rateper target per minute is shown below the image for each nuclease at eachconcentration of probe and target tested.

It was demonstrated in Example 32 that the use of the Pfu FEN-1structure-specific nuclease in the INVADER-directed cleavage reactionresulted in a faster rate of product accumulation than did the use ofthe CLEAVASE A/G. The data presented here demonstrates that the use ofMja FEN-1 nuclease with the fluorescein labeled probe further increasesthe amount of product generated by an average of about 50%,demonstrating that, in addition to the Pfu FEN-1 nuclease, the Mja FEN-1nuclease is a preferred structure-specific nuclease for the detection ofnucleic acid targets by the method of the present invention.

Example 39 Detection of RNA Target Nucleic Acids Using Miniprobe andStacker Oligonucleotides

In addition to the detection of the M13 DNA target material describedabove, a miniprobe/stacker system was designed to detect the HCV-derivedRNA sequences described in Example 19. A probe of intermediate length,either a long mid-range or a short standard probe, was also tested. Theminiprobe used (oligo 42-168-1) has the sequence:5′-TET-CCGGTCGTCCTGG-3′ (SEQ ID NO:120), the stacker oligonucleotideused (oligo 32-085) with this miniprobe has the sequence:5′-CAATTCCGGTGTACTACCGGTTCC-3′ (SEQ ID NO:121). The slightly longerprobe, used without a stacker (oligo 42-088), has the sequence:5′-TET-CCGGTCGTCCTGGCAA-3′ (SEQ ID NO:122). The INVADER oligonucleotideused with both probes has the sequence: 5′-GTTTATCCAAGAAAGGACCCGGTC-3′(SEQ ID NO:47). The reactions included 50 fmole of target RNA, 10 pmoleof the INVADER oligonucleotide and 5 pmole of the miniprobeoligonucleotide in 10 μl of buffer containing 10 mM MES, pH 6.5 with 150mM LiCl, 4 mM MnCl₂, 0.05% each Tween-20 and NP-40, and 39 units ofRNAsin (Promega). When used, 10 pmoles of the stacker oligonucleotidewas added. These components were combined, overlaid with CHILLOUTevaporation barrier, and warmed to 50° C.; the reactions were started bythe addition of 5 polymerase units of DNAPTth, to a final reactionvolume of 10 μl. After 30 minutes at 50° C., reactions were stopped bythe addition of 8 μl of 95% formamide, 10 mM EDTA and 0.02% methylviolet. The samples were heated to 90° C. for 1 minute and 2.5 μl ofeach of these reactions were resolved by electrophoresis through a 20%denaturing polyacrylarnide (19:1 cross link) with 7M urea in a buffer of45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA, and the labeled reactionproducts were visualized using the FMBIO-100 Image Analyzer (Hitachi).The resulting image is shown in FIG. 84.

In FIG. 84, lanes 1 and 2 show the products of reactions containing theHCV INVADER oligonucleotide and the longer probe (oligo 42-088), withoutand with the target RNA present, respectively. Lanes 3, 4, and 5 showthe products of reactions containing the INVADER oligonucleotide and theshorter probe (oligo 42-168-1). Lane 3 is a control reaction withouttarget RNA present, while lanes 4 and 5 have the target, but are withoutor with the stacker oligonucleotide, respectively.

Under these conditions the slightly longer (16 nt) probe oligonucleotidewas cleaved quite easily without the help of a stacker oligonucleotide.In contrast, the shorter probe (13 nt) required the presence of thestacker oligonucleotide to produce detectable levels of cleavage. Thesedata show that the miniprobe system of target detection byINVADER-directed cleavage is equally applicable to the detection of RNAand DNA targets. In addition, the comparison of the cleavage performanceof longer and shorter probes in the absence of a stacker oligonucleotidegive one example of the distinction between the performance of theminiprobe/stacker system and the performance of the mid-range and longprobes in the detection of nucleic acid targets.

Example 40 Effect of an Unpaired 3′ Tail on Transcription From AComplete (Un-Nicked) Promoter

In designing the method of transcription-based visualization of theproducts of INVADER-directed cleavage, it was first necessary to assessthe effect of a 3′ tail on the efficiency of transcription from a fulllength promoter. The duplexes tested in this Example are shown at thebottom of FIG. 93, and are shown schematically in FIGS. 85A-C.

Transcription reactions were performed using the MEGAshortscript™ systemfrom Ambion, Inc. (Austin, Tex.), in accordance with the manufacturer'sinstructions with the exception that a fluorescein labeledribonucleotide was added. Each DNA sample was assembled in 4 μL ofRNAse-free dH₂O. Reactions 1-3 each contained 10 pmole of the copytemplate oligo 150 (SEQ ID NO:123); reaction 2 contained 10 pmole of thepromoter oligo 151 (SEQ ID NO:124); sample 3 contained 10 pmole of the3′ tailed promoter oligo 073-065 (SEQ ID NO:125); sample 4 had no addedDNA. To each sample, 6 μl of a solution containing 1 μl of 10×Transcription Buffer, 7.5 mM each rNTP, 0.125 mM fluorescein-12-UTP(Boehringer) and 1 μl T7 MEGAshortscript™ Enzyme Mix was added. Thesamples were then incubated at 37° C. for 1 hour. One microliter ofRNase-free DNase 1 (2 U/μl) was added to each sample and the sampleswere incubated an additional 15 minutes at 37° C. The reactions werethen stopped by the addition of 10 μl of a solution of 95% formamide, 5mM Na₂EDTA, with loading dyes. All samples were heated to 95° C. for 2minutes and 4 μl of each sample were resolved by electrophoresis througha 20% denaturing acrylamide gel (19:1 cross-linked) with 7M urea, in abuffer containing 45 mM Tris-Borate (pH 8.3), 1.4 mM EDTA. The gel wasanalyzed with a FMBIO II fluorescence image analyzer, and the resultingimage is shown in FIG. 93. The RNA produced by successful transcriptionappears near the middle of the panel, as indicated (“RNA”).

Examination of the products of transcription shown in lanes 2 and 3 showthat the presence of the 3′ tail on the full-length promoter has anadverse affect on the efficiency of transcription, but does not shut itoff completely. Because the objective of the transcription-basedvisualization assays of the present invention is to discriminate betweenuncleaved probe and the shorter products of the invasive cleavage assay(cut probe), these data indicate that production of a full-lengthpromoter in the cleavage reaction would be difficult to resolve from thebackground created by transcription from promoters containing theuncleaved probe if no other oligonucleotides were included in the assay.Means of suppressing transcription from such a branched promoter arediscussed in the Description of the Invention and discussed below in Ex.43.

Example 41 Examination of the Influence of the Position of the Nick onthe Efficiency of Transcription From Partial and Complete CompositeBacteriophage T7 Promoters

In the Description of the Invention, the procedure for testingprospective promoter pieces for suitability in an invasivecleavage-linked assay is described. One aspect of the test is to examinethe effect a chosen nick site has on the efficiency of transcriptionfrom the final composite promoter. In addition, the individual pieces ofnicked promoter are tested for transcription activity in the presence ofthe full-length un-nicked strand. In this experiment, a comparison onthese points is made between a composite promoter having a nick in thenon-template strand between nucleotides −11 and −10 relative to theinitiation site (+1), and a promoter having a nick on the same strand,but positioned between nucleotides −8 and −7. The Figure numbers for theschematic representations of the contents of each reaction are indicatedbelow each lane (e.g., 85A=FIG. 85A). The site where the nick would bein a fully assembled composite promoter using the reactionoligonucleotides is also indicated below each lane (“−11/−10” and“−8/−7”).

Transcription reactions were performed using the MEGAshortscript™system, in accordance with the manufacturer's instructions, but with theexception that a fluorescein labeled ribonucleotide was added. Each DNAsample was assembled in 4 μl of RNAse-free dH₂O. Reaction 1 had no addedDNA. Reactions 2-9 each contained 10 pmole of the copy template oligo150 (SEQ ID NO:123). Reactions 3 and 4 contained 10 pmole of the −11“cut” probe (oligo 073-061-01; SEQ ID NO:127) or 20 pmole of the −10partial promoter oligo 073-061-02 (SEQ ID NO:130), respectively, andreaction 5 contained both. Reactions 6 and 7 contained either the 10pmole of the −8 “cut” probe (oligo 073-062-01; SEQ ID NO:126) or 20pmoles of the −7 partial promoter oligo 073-062-02 (SEQ ID NO:129),respectively, and reaction 8 contained them both. Reaction 9 contained10 pmole of the intact promoter oligo 151 (SEQ ID NO:124).

The transcription reactions were initiated, incubated, terminated andthe reaction products were resolved and imaged as described in Ex. 40.The resulting image is shown in FIG. 92. The reaction numbers correspondto the lane numbers above the image. The RNA created by successfultranscription appears in the upper third of the image. Comparison to thepositive control reaction (rxn. 9) shows that the full-length RNAproduced by each of the composite promoters is the same size as thatproduced in the control reaction, indicated that transcription initiatedat the same site in each reaction.

In FIG. 92, lanes 3, 4, and 5 compare transcription from the two speciesof partially assembled promoters (see schematics in FIGS. 86A and B) andthe fully assembled composite promoter (FIG. 88B) having a nick betweennucleotides −11 and −10 relative to the start of transcription. It canbe seen from these data that neither partial promoter (lanes 3 and 4) isable to support transcription of the copy template, but that thecomposite promoter (lane 5) with this nick site is strongly transcribed.Surprisingly, comparison to the control reaction (lane 9) shows that thepresence of a nick at this site (−11/−10) actually enhancestranscription. While not limiting the present invention to anyparticular mechanism, it is believed that the enhancement oftranscription is a result of both suppressing the formation of theshorter abortive transcripts and by allowing greater accumulation of thefull length product. This result is highly reproducible.

In FIG. 92, lanes 6, 7, and 8 compare transcription a similar set ofpartial and complete promoters in which the nick is shifted 3 residuescloser to the transcription start site. Examination of lane 6 shows thatthe presence of 3 extra bases on the −8“cut” probe (compared to the −11“cut” probe in lane 3) allow this partial promoter to initiatetranscription. This indicates that the −8/−7 site would be a poor choicefor use in this embodiment of the present invention.

This experiment demonstrates the process for determining the suitableplacement of a nick within a promoter assembly to achieve the desiredresult. Similar tests can easily be designed for testing other nickswithin the bacteriophage T7 promoter tested in this Example, or fortesting suitable nick placement in any desired phage, prokaryotic oreukaryotic promoter.

Example 42 Detection of the Products of INVADER-Directed CleavageThrough Transcription from a Composite Promoter

The Examples described above indicate that a small oligonucleotide canbe used to complete assembly of a composite T7 promoter, therebyenabling transcription from that promoter. Earlier Examples demonstratethat the invasive cleavage reaction can be used release specific smalloligonucleotide products from longer probe oligonucleotides. In thisExample, it is demonstrated that these two observations can be combined,and that the products of the invasive cleavage reaction can be used tocomplete a promoter and enable subsequent transcription. The schematicrepresentations of the composite promoters tested in this Example areshown in FIG. 88.

Two invasive cleavage reactions were set up, one without (rxn. 1) andone with (rxn. 2) input target DNA. The reactions (1 and 2) comprised 10mM MOPS (pH 7.5), 0.05% Tween-20, 0.05% NP-40 and 20 pmoles probe oligo073-067-01 (SEQ ID NO:132) and 10 pmoles INVADER oligo 073-073-02 (SEQID NO:134) in a volume of 14 μl. Reaction 2 also included 100 fmolesM13mp18 ssDNA. The samples were placed at 60° C. and 6 μl of a solutioncontaining 20 ng of Mja FEN-1 and 40 mM Mg₂Cl were added to each sampleto start the reactions. The samples were incubated at 60° C. for 30minutes and stopped by the addition of 3 μl of 2.5M NaOAc, 83 mM Na₂EDTA(pH 8.0). Each sample was transferred to a 1.5 ml microcentrifuge tubeand then the DNAs were precipitated by the addition of 60 μl of chilled100% ethanol, and were stored at −20° C. for 20 minutes. The pelletswere collected by microcentrifugation, washed once with 80% ethanol toremove excess salt, then dried under vacuum. The product of thisinvasive cleavage reaction is a 12 nt oligonucleotide having thesequence: 5′-CGAAATTAATAC-3′ (SEQ ID NO:128), termed the −12 cut probe(same sequence as oligo 073-073-03).

For transcription, the dried samples were each dissolved in 4 μl of asolution containing 1 pmole copy template oligo 150 and 2 pmoles −11partial promoter oligo 073-073-012 (SEQ ID NO:131). Control samples 3and 4 each contained 1 pmole of the copy template oligo 150; sample 3also contained 1 pmole probe oligo 073-067-01 (SEQ ID NO:132) and 2pmoles −11 partial promoter oligo 073-073-012 (see structure 88A);sample 4 contained 1 pmole −12 “cut” probe oligo 073-073-03 (SEQ IDNO:128) and 2 pmoles −11 partial promoter oligo 073-073-012 (seestructure 88B). These are the structures that would be expected to existin the transcription reactions from the two invasive cleavage reactionsdescribed above.

The transcription reactions were initiated, incubated, terminated andthe products were resolved and imaged as described in Ex. 40. Theresulting image is shown in the right half of FIG. 89 (lanes 6-9).Samples 3 and 4 appear in lanes 6 and 7, respectively, and the reactions1 and 2 from the invasive cleavage reaction products (indicated by theuse of the lower case “i”), appear in lanes 8 and 9, respectively. Thenumber of the Fig. showing the schematic representation of the expectedpromoter structure in each reaction is indicated above each lane, andthe placement of the nick is also indicated. The uppercase lettersindicate which structure in the particular Figure to examine for eachreaction. The lowercase “i” above lanes 8 and 9 indicate that thesetranscriptions were derived from actual invasive cleavage reactions.These products are compared to the RNA produced in the control reactionin lane 5, the procedure for which is described in Ex. 44. The RNAcreated by successful transcription appears in the upper third of thepanel (indicated by “RNA”).

The reaction shown in lane 6 shows no transcription. This demonstratesthat a nick between nucleotides −12 and −11 in the on-template strand ofthe T7 promoter eliminates transcription if the promoter is assembledfrom uncut probe such as the 3′ end of the probe forms a branch withinthe promoter sequence. This is in contrast tot he results seen with the−11/−10 nick examined below. Further, the transcript apparent in lane 7shows that an unbranched promoter with a nick at the same site (−12/−11)produces the correct RNA, with few abortive initiation products (seelanes 2 and 5 of FIG. 89, described in Ex. 44). The reactions in lanes 8and 9 demonstrate that the same effect is observed when the invasivecleavage reaction is the sole source of the upstream piece (−12 cutprobe) of the T7 promoter. It is worthy of note that the promoter thatis transcribed in lane 8 is made complete by the presence of 1 pmole ofa synthetic “cut” probe oligo, without any uncut probe in the mixture,while the promoter that is transcribed in lane 9 is completed by theproduct of an invasive cleavage reaction that had only 100 fmole oftarget DNA in it. This reaction also included the residual uncut probe(up to approx. 10 pmoles), which may compete for binding at the samesite. Nonetheless, the transcriptions from the invasive cleavagereaction products are only slightly reduced in efficiency, and are justas free of background as is the “no target” sample (lane 8). ThisExample clearly demonstrates that the cleavage products from theinvasive cleavage reaction can be used in combination with a partialpromoter oligo to promote the production of RNA, without backgroundtranscription generated by the presence of the uncut probe. This RNAproduct is clearly dependent on the presence of the target material inthe invasive cleavage reaction.

Example 43 Shutting Down Transcription From a “Leaky” Branched T7Composite Promoter Through the Use of a Downstream Partial PromoterOligonucleotide Having a 5′ Tail

The previous Example demonstrated that placement of a nick in thenon-template strand of a bacteriophage T7 promoter between the −12 and−11 nucleotides, relative to the transcription start site, preventstranscription of the branched promoter while allowing transcription whenthe composite promoter is assembled using the cut probe. When the nickis placed in other locations in the T7 promoter, transcription may beinitiated from either promoter, although it is usually less efficientfrom the branched promoter. This Example demonstrates that the additionof a 5′ tail that can base pair to the uncut probe (FIG. 90A) to thedownstream partial promoter piece effectively blocks transcription fromthat promoter, but does not prevent transcription when a cut probecompletes the promoter (FIG. 90B).

Two invasive cleavage reactions were set up, one without (rxn. 7) andone with (rxn. 8) input target DNA. The reactions (7 and 8) comprisedIOmmM MOPS (pH 7.5), 0.05% Tween-20, 0.05% NP-40 and 20 pmoles probeoligo 073-067-01 (SEQ ID NO:132)and 10 pmoles INVADER oligo 073-067-02(SEQ ID NO:133) in a volume of 14 μl. Reaction 8 also included 100fmoles M13mp18 ssDNA. The samples were placed at 60° C. and 6 μl of asolution containing 20 ng of Mja FEN-1 and 40 mM Mg₂Cl were added toeach sample to start the reactions. The samples were incubated at 60° C.for 30 minutes and then stopped by the addition of 3 μl of 2.5M NaOAc,83 mM Na₂EDTA (pH.8.0). Each sample was transferred to a 1.5 mlmicrocentrifuge tube and the DNAs were precipitated, washed and dried asdescribed in Ex. 42. The product of this invasive cleavage reaction is13 nt oligonucleotide sequence, 5′-CGAAATTAATACG-3′ (SEQ ID NO:127),termed the −11 cut probe (same sequence as oligo 073-061-01 which isreferred to as the −11 “cut” probe to indicate it was not generated inan invasive cleavage reaction).

In the transcription reactions, all of the DNAs were dissolved in 4 μlof RNase-free dH₂O. Sample 1 had no added DNA, samples 2-8 contained 1pmole of the copy template oligo 150 (SEQ ID NO:123). In addition,sample 3 contained 1 pmole of −11 “cut” probe oligo 073-061-01 (SEQ IDNO:127) and 2 pmoles of −10 partial promoter oligo 073-061-02 (SEQ IDNO:130), sample 4 contained 1 pmole of probe oligo 073-067-01 and 2pmoles of −10 partial promoter oligo 073-061-02. Control sample 5contained 1 pmole of probe oligo 073-067-01 and 2 pmoles of partialpromoter w/5′ tail oligo 073-074(5′-TACTGACTCACTATAGGGTCTTCTATGGAGGTC-3′ (SEQ ID NO:146) (see structurein FIG. 90A) and sample 6 contained 1 pmole of −11 “cut” probe oligo073-061-01 and 2 pmoles of partial promoter w/5′ tail oligo 073-074 (seestructure in FIG. 90B). These are the structures (i.e., 90A and 90B)that would be expected to exist in the transcription reactions from thetwo invasive cleavage reactions described above.

The dried samples 7 and 8 from the invasive cleavage (above) were eachdissolved in 4 μl of dH₂O containing 1 pmole copy template oligo 150 and2 pmoles partial promoter w/5′ tail oligo 073-074. The transcriptionreactions were initiated, incubated, terminated and the reactionproducts were resolved and imaged as described in Ex. 40. The resultingimage is shown in FIG. 91.

In FIG. 91 the lane numbers correspond to the sample numbers; the numberof the Figure showing the schematic representation of the expectedpromoter structure in each reaction is indicated above each lane (“88”and “90”), and the placement of the nick is also indicated (“−11/−10”).The upper-case letters indicate which structure in the particular Figureto examine for each reaction. The lower case “i” above lanes 7 and 8indicates that these transcriptions were derived from actual invasivecleavage reactions. The RNA created by successful transcription appearsin the upper third of the panel, as indicated (“RNA”).

The control reactions in lanes 1 and 2, having either no DNA or havingthe only the copy template, produced no RNA as expected. The product inlane 4 demonstrates that the branched T7 promoter with a nick in thenon-template strand between nucleotides −11 and −10 can supporttranscription, albeit not as efficiently as the un-branched promoterwith the nick at the same site (lane 3). Examination of lane 5 showsthat the use of a partial promoter oligonucleotide with a short 5′ tailthat can basepair to the uncut probe as depicted in FIG. 90A,effectively suppresses this transcription but allows transcription whenthe probe does not have a 3′ tail (lane 6; schematic FIG. 90B). Thereactions in lanes 7 and 8 demonstrate that the same effect as observedwhen the invasive cleavage reaction is the sole source of the upstreampiece (−11 cut probe, SEQ ID NO:127) of the T7 promoter. It is worthy ofnote that the promoter that is transcribed in sample 6 is made completeby the presence of 1 pmole of a synthetic “cut probe”, without any uncutprobe in the mixture, while the promoter that is transcribed in sample 8is completed by the product of an invasive cleavage reaction that hadonly 100 fmole of target DNA in it. This reaction also included theresidual uncut probe (up to approximately 19 pmoles), which may competefor binding at the same site. Nonetheless, the transcriptions from theinvasive cleavage reaction products are just as strong and just as freeof background in the “no target” samples.

This Example clearly demonstrates that the cleavage products from theinvasive cleavage reaction can be used in combination with a partialpromoter oligonucleotide having a 5′ tail to promote the production ofRNA, without background transcription generated by the uncut probe. ThisRNA product is clearly dependent on the presence of the target materialin the invasive cleavage reaction.

Example 44 Creation of a Complete Bacteriophage T7 Promoter by DNAPolymerase-Mediated Extension of a Cut Probe Comprising a Partial T7Promoter

As demonstrated in the Examples above, transcription cannot occur fromthe T7 promoter unless a complete promoter region is present. In theabove Examples, a complete promoter containing a nick in one strand wascreated by annealing a cut probe generated from an invasive cleavagereaction to a copy template that was annealed to a partial promoteroligo. An alternative means of creating a complete promoter in a mannerdependent upon detection of a target sequence in an invasive cleavagereaction is to anneal the cut probe to a copy template devoid of apartial promoter oligo. The 3′-OH present at the end of the annealed cutprobe is then extended by a DNA polymerase to create a complete andun-nicked promoter that is transcription-competent.

In this Example, the promoter was made complete through the use ofprimer extension, rather that by the co-hybridization of anotheroligonucleotide. The reaction steps are diagrammed schematically in FIG.87. Two invasive cleavage reactions were set up, one without (rxn. 1)and one with (rxn. 2) input target DNA. The reactions (1 and 2)comprised 10 mM MOPS (pH 7.5), 0.05% Tween-20, 0.05% NP-40 and 20 pmolesprobe oligo 073-067-01 (SEQ ID NO:132) and 10 pmoles INVADER oligo073-073-02 (SEQ ID NO:134) in a volume of 14 μl. Reaction 2 alsoincluded 100 fmoles M13mp18 ssDNA. The samples were placed at 60° C. and6 μl of a solution containing 20 ng of Mja FEN-1 and 40 mM Mg₂Cl wereadded to each sample to start the reactions. The samples were incubatedat 60° C. for 30 minutes and stopped by the addition of 3 μl of 2.5MNaOAc, 83 mM Na₂EDTA (pH 8.0). Each sample was transferred to a 1.5 mlmicrocentrifuge tube and then the DNAs were precipitated, washed anddried as described in Ex. 42. The product of this invasive cleavagereaction is the 12 nt oligonucleotide sequence: 5′-CGAAATTAATAC-3′ (SEQID NO:128), termed the −12 cut probe (same sequence as oligo 073-073-03which is referred to as the −12 “cut” probe to indicate it was notgenerated in an invasive cleavage reaction).

To allow extension of these products using a template-dependent DNApolymerase, a 20 μl solution containing 20 mM Tris-HCl (pH 8.5), 1.5 mMMg₂Cl, 50 mM KCl, 0.05% Tween-20, 0.05% NP-40, 25 μM each dNTP, 0.25units Taq DNA polymerase (Boehringer) and 2 μM copy template oligo 150(SEQ ID NO:123) was added to each of the dried cleavage samples. Thesamples were incubated at 30° C. for 1 hr. The primer extensionreactions were stopped by the addition of 3 μl of 2.5M NaOAc with 83 mMNa₂EDTA (pH 8.0)/sample. Each sample was transferred to a 1.5 mlmicrocentrifuge tube and the DNAs were precipitated, washed and dried asdescribed in Ex. 42.

Samples 1 and 2 were then dissolved in 4 μl RNase-free dH₂O. Samples 3,4 and 5 are control reactions: sample 3 was 4 μl of RNase-free dH₂Owithout added DNA, sample 4 contained 1 pmole of the copy template oligo150 (SEQ ID NO:123) in 4 μl of RNase-free dH₂O, and sample 5 contained 1pmole of the same copy template and 1 pmole of the complete promoteroligo 151 (SEQ ID NO:124) in RNase-free dH₂O.

Transcription reactions were performed using the MEGAshortscript™system, in accordance with the manufacturer's instructions, but with theaddition of a fluorescein labeled ribonucleotide. To each sample, 6 μlof a solution containing 1 μl of 10× Transcription Buffer, 7.5 mM eachrNTP, 0.125 mM fluorescein-12-UTP (Boehringer) and 1 μl T7MEGAshortscript™ Enzyme Mix was added. The samples were incubated at 37°C. for 1 hour. One μl of RNase-free DNase 1 (2 U/μl) was added to eachsample and they were incubated an additional 15 minutes at 37° C. Thereactions were stopped by the addition of 10 μl of a solution of 95%formamide, 5 mM NaEDTA, with loading dyes. All samples were heated to95° C. for 2 minutes and four μl of each sample were resolved byelectrophoresis through a 20% denaturing acrylamide gel (19:1cross-linked) with 7 M urea, in a buffer containing 45 mM Tris-Borate(pH 8.3), 1.4 mM EDTA. The results were imaged using the MolecularDynamics Fluoroimager 595, with excitation at 488 nm and, emissiondetected at 530 nm.

The resulting image is shown in lanes 1 through 5 of FIG. 89; the lanenumbers correspond to the sample numbers. The Figure numberscorresponding to the schematic representations of the promoterstranscribed in each reaction as indicated above the lanes. The RNAproduct from successful transcription appears in the upper third of thepanel, as indicated (“RNA”). Unincorporated labeled nucleotide appearsas a dense signal near the bottom (“NTPs”). Short transcription productscaused by aborted initiation events (Milligan and Uhlenbeck, MethodsEnzymol., 180:51 [1989]) appear as bands just above the free nucleotidein the lanes showing active transcription (i.e., lanes 2 and 5).

It can clearly be seen from the data in lanes 1 and 2 that thetranscription is dependent on the presence of the target material in theinvasive cleavage reaction. It is shown elsewhere (see lane 3, FIG. 92)that the product of the cleavage reaction is not in itself sufficient toallow transcription from the copy template. Thus, the action of the DNApolymerase in extending the hybridized cut probe across the promoter isa necessary step in enabling the transcription in this embodiment. Thesedata clearly demonstrate that both template-dependent extension by DNApolymerase, and extension followed by transcription are suitable methodsof visualizing the products of the invasive cleavage assay. As discussedin the Description of the Invention, the products of thermal breakdownthat possess 3′ terminal phosphates would not be extended, and wouldthus be precluded from contributing to background transcription.

Example 45 Test For The Dependence of an Enzyme on The Presence of anUpstream Oligonucleotide

When choosing a structure-specific nuclease for use in a sequentialinvasive cleavage reaction it is preferable that the enzyme have littleability to cleave a probe 1) in the absence of an upstreamoligonucleotide, and 2) in the absence of overlap between the upstreamoligonucleotide and the downstream labeled probe oligonucleotide. FIGS.99 a-e depicts the several structures that can be used to examine theactivity of an enzyme that is confronted with each of these types ofstructures. The structure a (FIG. 99 a) shows the alignment of a probeoligonucleotide with a target site on bacteriophage M13 DNA (M13sequences shown in FIG. 99 are provided in SEQ ID NO:163) in the absenceof an upstream oligonucleotide. Structure b (FIG. 99 b) is provided withan upstream oligonucleotide that does not contain a region of overlapwith the labeled probe (the label is indicated by the star). Instructures c, d and e (FIGS. 99 c-e) the upstream oligonucleotides haveoverlaps of 1, 3 or 5 nucleotides, respectively, with the downstreamprobe oligonucleotide and each of these structures represents a suitableinvasive cleavage structure. The enzyme Pfu FEN-1 was tested foractivity on each of these structures and all reactions were performed induplicate.

Each reaction comprised 1 μM 5′ TET labeled probe oligonucleotide89-15-1 (SEQ ID NO:152), 50 nM upstream oligonucleotide (either oligo81-69-2 [SEQ ID NO:153], oligo 81-69-3 [SEQ ID NO:154], oligo 81-69-4[SEQ ID NO:155], oligo 81-69-5 [SEQ ID NO:156], or no upstreamoligonucleotide), 1 fmol M13 target DNA, 10 mg/ml tRNA and 10 ng of PfuFEN-1 in 10 μl of 10 mM MOPS (pH 7.5), 7.5 mM MgCl₂ with 0.05% each ofTween 20 and Nonidet P-40.

All of the components except the enzyme and the MgCl₂ were assembled ina final volume of 8 μl and were overlaid with 10 μl of Chill-Out™ liquidwax. The samples were heated to the reaction temperature of 69° C. Thereactions were started by the addition of the Pfu FEN-1 and MgCl₂, in a2 μl volume. After incubation at 69° C. for 30 minutes, the reactionswere stopped with 10 μl of 95% formamide, 10 mM EDTA, 0.02% methylviolet. Samples were heated to 90° C. for 1 min immediately beforeelectrophoresis through a 20% denaturing acrylamide gel (19:1cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3,1.4 mM EDTA. Gels were then analyzed with a FMBIO-100 Hitachi FMBIOfluorescence imager. The resulting image is displayed in FIG. 100.

In FIG. 100, lanes labeled “a” contain the products generated fromreactions conducted without an upstream oligonucleotide (structure a),lanes labeled “b” contain an upstream oligonucleotide that does notinvade the probe/target duplex (structure b). Lanes labeled “c”, “d” and“e” contain the products generated from reactions conducted using anupstream oligonucleotide that invades the probe/target duplex by 1, 3 or5 bases, respectively. The size (in nucleotides) of the uncleaved probeand the cleavage products is indicated to the left of the image in FIG.100.

As shown in FIG. 100, cleavage of the probe was not detectable whenstructures a and b were utilized. In contrast, cleavage products weregenerated when invasive cleavage structures were utilized (structuresc-e). These data show that the Pfu FEN-1 enzyme requires an overlappingupstream oligonucleotide for specific cleavage of the probe.

Any enzyme may be examined for its suitability for use in a sequentialinvasive cleavage reaction by examining the ability of the test enzymeto cleave structures a-e (it is understood by those in the art that thespecific oligonucleotide sequences shown in FIGS. 99 a-e need not beemployed in the test reactions; these structures are merely illustrativeof suitable test structures). Desirable enzymes display little or nocleavage of structures a and b and display specific cleavage ofstructures c-e (i.e., they generate cleavage products of the sizeexpected from the degree of overlap between the two oligonucleotidesemployed to form the invasive cleavage structure).

Example 46 Use of the Products of a First Invasive Cleavage Reaction toEnable a Second Invasive Cleavage Reaction With a Net Gain inSensitivity

As discussed in the Description of The Invention above, the detectionsensitivity of the invasive cleavage reaction can be increased by theperforming a second round of invasive cleavage using the products of thefirst reaction to complete the cleavage structure in the second reaction(shown schematically in FIG. 96). In this Example, the use of a probethat, when cleaved in a first invasive cleavage reaction, forms anintegrated INVADER oligo and target molecule for use in a secondinvasive cleavage reaction, is illustrated (shown schematically in FIG.97).

A first probe was designed to contain some internal complementarity sothat when cleaved in a first invasive cleavage reaction the product(“Cut Probe 1”) could form a target strand comprising an integralINVADER oligonucleotide, as depicted in FIG. 97. A second probe wasprovided in the reaction that would be cleaved at the intended site whenhybridized to the newly formed target/INVADER (FIG. 97). To demonstratethe gain in signal due to the performance of sequential invasivecleavages, a standard invasive cleavage assay, as described above, wasperformed in parallel.

All reactions were performed in duplicate. Each standard (i.e.,non-sequential) invasive cleavage reaction comprised 1 μM 5′fluorescein-labeled probe oligo 073-182(5′F1-AGAAAGGAAGGGAAGAAAGCGAA-3′; SEQ ID NO:157), 10 nM upstream oligo81-69-4 (5′-CTTGACGGGGAAAGCCGGCGAACGTGGCGA-3′; SEQ ID NO:155), 10 to 100attomoles of M13 target DNA, 10 mg/ml tRNA and 10 ng of Pfu FEN-1 in 10μl of 10 mM MOPS (pH 7.5), 8 mM MgCl₂ with 0.05% each of Tween 20 andNonidet P-40. All of the components except the enzyme and the MgCl₂ wereassembled in a volume of 7 μl and were overlaid with 10 μl of Chill-Out™liquid wax. The samples were heated to the reaction temperature of 62°C. The reactions were started by the addition of the Pfu FEN-1 andMgCl₂, in a 2 μl volume. After incubation at 62° C. for 30 minutes, thereactions were stopped with 10 μl of 95% formamide, 10 mM EDTA, 0.02%methyl violet.

Each sequential invasive cleavage reaction comprised 1 μM 5′fluorescein-labeled oligonucleotide 073-191 (the first probe or “Probe1”, 5′F1-TGGAGGTCAAAACATCG ATAAGTCGAAGAAAGGAAGGGAAGAAAT-3′; SEQ IDNO:158), 10 nM upstream oligonucleotide 81-69-4 (5′-CTTGACGGGGAAAGCCGGCGAACGTGGCGA-3′; SEQ ID NO:155), 1 μM of 5′fluorescein labeled oligonucleotide 106-32 (the second probe or “Probe2”, 5′F1-TGTTTTGACCTCCA-3′; SEQ ID NO:159), 1 to 100 amol of M13 targetDNA, 10 mg/ml tRNA and 10 ng of Pfu FEN-1 in 10 μl of 10 mM MOPS (pH7.5), 8 mM MgCl₂ with 0.05% each of Tween 20 and Nonidet P-40. All ofthe components except the enzyme and the MgCl₂ were assembled in avolume of 8 μl and were overlaid with 10 μl of Chill-Out™ liquid wax.The samples were heated to the reaction temperature of 62° C. (thistemperature is the optimum temperature for annealing of Probe 1 to thefirst target). The reactions were started by the addition of Pfu FEN-1and MgCl₂, in a 2 μl volume. After incubation at 62° C. for 15 minutes,the temperature was lowered to 58° C. (this temperature is the optimumtemperature for annealing of Probe 2 to the second target) and thesamples were incubated for another 15 min. Reactions were stopped by theaddition of 10 μl of 95% formamide, 20 mM EDTA, 0.02% methyl violet.

Samples from both the standard and the sequential invasive cleavagereactions were heated to 90° C. for 1 min immediately beforeelectrophoresis through a 20% denaturing acrylamide gel (19:1cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3,1.4 mM EDTA. The gel was then analyzed with a Molecular DynamicsFluoroImager 595. The resulting image is displayed in FIG. 101 a. Agraph showing measure of fluorescence intensity for each of the productbands is shown in FIG. 101 b.

In FIG. 101 a, lanes 1-5 contain the products generated in standardinvasive cleavage reactions that contained either no target (lane 1), 10amol of target (lanes 2 and 3) or with 100 amol of target (lanes 4 and5). The uncleaved probe is seen as a dark band in each lane about halfway down the panel and the cleavage products appear as a smaller blackband near the bottom of the panel, the position of the cleavage productis indicated by an arrow head to the left of FIG. 101 a. The gray ladderof bands seen in lanes 1-5 is due to the thermal degradation of theprobe as discussed above and is not related to the presence or absenceof the target DNA. The remaining lanes display products generated insequential invasive cleavage reactions that contained 1 amol of target(lanes 6 and 7), 10 amol of target (lanes 8 and 9) and 100 amol oftarget (lanes 10 and 11). The uncleaved first probe (Probe 1; labeled “1uncut”) is seen near the top of the panel, while the cleaved first probeis indicated as “1: cut”. Similarly, the uncleaved and cleaved secondprobe are indicated as “2: uncut” and 2: cut,” respectively.

The graph shown in FIG. 101 b compares the amount of product generatedfrom the standard reaction (“Series 1”) to the amount of productgenerated from the second step of the sequential reaction (“Series 2”).The level of background fluorescence measured from a reaction thatlacked target DNA was subtracted from each measurement. It can be seenfrom the table located below the graph that the signal from the standardinvasive cleavage assay that contained 100 attomoles of target DNA wasnearly identical to the signal from the sequential invasive cleavageassay in which 1 attomole of target was present, indicating that theinclusion of a second cleavage structure increases the sensitivity ofthe assay 100 to 200-fold. This boost in signal allows easy detection oftarget nucleic acids at the sub attomole level using the sequentialinvasive cleavage assay, while the standard assay, when performed usingthis enzyme for only 30 minutes, does not generate detectable product inthe presence of 10 attomoles of target.

When the amount of target was decreased by 10 or 100 fold in thesequential invasive cleavage assay, the intensity of the signal wasdecreased by the same proportion. This indicates that the quantitativecapability of the invasive cleavage assay is retained even whenreactions are performed in series, thus providing a nucleic aciddetection method that is both sensitive and quantitative.

While in this Example, the two probes used had different optimalhybridization temperatures (i.e., the temperature empirically determinedto give the greatest turnover rate in the given reaction conditions),the probes may also be selected (i.e., designed) to have the sameoptimal hybridization temperature so that a temperature shift duringincubation is not necessary.

Example 47 The Products of a Completed Sequential Invasive CleavageReaction Cannot Cross Contaminate Subsequent Similar Reactions

As discussed in the Description of the Invention, the serial nature ofthe multiple invasive cleavage events that occur in the sequentialinvasive cleavage reaction, in contrast to the reciprocating nature ofthe polymerase chain reaction and similar doubling assays, means thatthe sequential invasive cleavage reaction is not subject tocontamination by the products of like reactions because the products ofthe first cleavage reaction do not participate in the generation of newsignal in the second cleavage reaction. If a large amount of a completedreaction were to be added to a newly assembled reaction, the backgroundthat would be produced would come from the amount of target that wasalso carried in, combined with the amount of already-cleaved probe thatwas carried in. In this Example, it is demonstrated that a very largeportion of a primary reaction must be introduced into the secondaryreaction to create significant signal.

A first or primary sequential invasive cleavage reaction was performedas described above using 100 amol of target DNA. A second set of 5reactions were assembled as described in Ex. 46 with the exception thatportions of the first reaction were introduced and no additional targetDNA was included. These secondary reactions were initiated and incubatedas described above, and included 0, 0.01, 0.1, 1, or 10% of the firstreaction material. A control reaction including 100 amol of target wasincluded in the second set also. The reactions were stopped, resolved byelectrophoresis and visualized as described above, and the resultingimage is displayed in FIG. 102. The primary probe, uncut second probeand the cut 2nd probe are indicated on the left as “1: cut”, 2: uncut”and 2: cut”, respectively.

In FIG. 102, lane 1 shows the results of the first reaction with theaccumulated product at the bottom of the panel, and lane 2 show a 1:10dilution of the same reaction, to demonstrate the level of signal thatcould be expected from that level of contamination, without furtheramplification. Lanes 3 through 7 show the results of the secondarycleavage reactions that contained 0, 10, 1, 0.1 or 0.01% of the firstreaction material added as contaminant, respectively and lane 8 shows acontrol reaction that had 100 amol of target DNA added to verify theactivity of the system in the secondary reaction. The signal level inlane 4 is as would be expected when 10% of the pre-cleaved material istransferred (as in lane 2) and 10% of the transferred target materialfrom the lane 1 reaction is allowed to further amplify. At all levels offurther dilution the signal is not readily distinguished frombackground. These data demonstrate that while a large-scale transferfrom one reaction to another may be detectable, cross contamination bythe minute quantities that would be expected from aerosol or fromequipment contamination would not be easily mistaken for a falsepositive result. These data also demonstrate that when the products ofone reaction are deliberately carried over into a fresh sample, theseproducts do not participate in the new reaction, and thus do not affectthe level of target-dependent signal that may be generated in thatreaction.

Example 48 Detection of Human Cytomegalovirus Viral DNA by InvasiveCleavage

The previous Example demonstrates the ability of the invasive cleavagereaction to detect minute quantities of viral DNA in the presence ofhuman genomic DNA. In this Example, the probe and INVADERoligonucleotides were designed to target the 3104-3061 region of themajor immediate early gene of human cytomegalovirus (HCMV) as shown inFIG. 103. In FIG. 103, the INVADER oligo (89-44; SEQ ID NO:160) and thefluorescein (F1)-labeled probe oligo (89-76; SEQ ID NO:161) are shownannealed along a region of the HCMV genome corresponding to nucleotides3057-3110 of the viral DNA (SEQ ID NO:162). The probe used in thisExample is a poly-pyrimidine probe and as shown herein the use of apoly-pyrimidine probe reduces background signal generated by the thermalbreakage of probe oligos.

The genomic viral DNA was purchased from Advanced Biotechnologies,Incorporated (Columbia, Md.). The DNA was estimated (but not certified)by personnel at Advanced Biotechnologies to be at a concentration of 170amol (1×10⁸ copies) per microliter. The reactions were performed inquadruplicate. Each reaction comprised 1 μM 5′ fluorescein labeled probeoligonucleotide 89-76 (SEQ ID NO:161), 100 nM INVADER oligonucleotide89-44 (SEQ ID NO:160), 1 ng/ml human genomic DNA, and one of fiveconcentrations of target HCMV DNA in the amounts indicated above eachlane in FIG. 104, and 10 ng of Pfu FEN-1 in 10 μl of 10 mM MOPS (pH7.5), 6 mM MgCl₂ with 0.05% each of Tween 20 and Nonidet P-40. All ofthe components except the labeled probe, enzyme and MgCl₂ were assembledin a final volume of 7 μl and were overlaid with 10 μl of Chill-Out™liquid wax. The samples were heated to 95° C. for 5 min, then reduced to62° C. The reactions were started by the addition of probe, Pfu FEN-1and MgCl₂, in a 3 μl volume. After incubation at 62° C. for 60 minutes,the reactions were stopped with 10 μl of 95% formamide, 10 mM EDTA,0.02% methyl violet. Samples were heated to 90° C. for 1 min immediatelybefore electrophoresis through a 20% acrylamide gel (19:1 cross-linked),with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.Gels were then analyzed with a Molecular Dynamics FluorImager 595.

The resulting image is displayed in FIG. 104. The replicate reactionswere run in groups of four lanes with the target HCMV DNA content of thereactions indicated above each set of lanes (0-170 amol). The uncleavedprobe is seen in the upper third of the panel (“Uncut 89-76”) while thecleavage products are seen in the lower two-thirds of the panel (“Cut89-76”). It can be seen that the intensity of the accumulated cleavageproduct is proportional to the amount of the target DNA in the reaction.Furthermore, it can be clearly seen in reactions that did not containtarget DNA (“no target”) that the probe is not cleaved, even in abackground of human genomic DNA. While 10 ng of human genomic DNA wasincluded in each of the reactions shown in FIG. 104, inclusion ofgenomic DNA up to 200 ng has slight impact on the amount of productaccumulated. The data did not suggest that 200 ng per 10 μl of reactionmixture represented the maximum amount of genomic DNA that could betolerated without a significant reduction in signal accumulation. Forreference, this amount of DNA exceeds what might be found in 0.2 ml ofurine (a commonly tested amount for HCMV in neonates) and is equivalentto the amount that would be found in about 5 μl of whole blood.

These results demonstrate that the standard (i.e., non-sequential)invasive cleavage reaction is a sensitive, specific and reproduciblemeans of detecting viral DNA. It can also be seen from these data thatthe use of a poly-pyrimidine probe reduces the background from thermalbreakage of the probe, as discussed in Example 22. Detection of 1.7 amolof target is roughly equivalent to detection of 10⁶ copies of the virus.This is equivalent to the number of viral genomes that might be found in0.2 mls of urine from a congenitally infected neonate (10² to 10⁶ genomeequivalents per 0.2 mls; Stagno et al., J. Infect. Dis., 132:568[1975]). Use of the sequential invasive cleavage assay would permitdetection of even fewer viral DNA molecules, facilitating detection inblood (10¹ to 10⁵ viral particles per ml; Pector et al., J. Clin.Microbiol., 30:2359 [1992]), which carries a much larger amount ofheterologous DNA.

From the above it is clear that the invention provides reagents andmethods to permit the detection and characterization of nucleic acidsequences and variations in nucleic acid sequences. The INVADER-directedcleavage reaction and the sequential INVADER-directed cleavage reactionof the present invention provide ideal direct detection methods thatcombine the advantages of the direct detection assays (e.g., easyquantification and minimal risk of carry-over contamination) with thespecificity provided by a dual or tri oligonucleotide hybridizationassay.

As indicated in the Description of the Invention, the use of sequentialinvasive cleavage reactions can present the problem of residual uncutfirst, or primary, probe interacting with the secondary target, andeither competing with the cut probe for binding, or creating backgroundthrough low level cleavage of the resulting structure. This is showndiagrammatically in FIGS. 105 and 106. In FIG. 105, the reactiondepicted makes use of the cleavage product from the first cleavagestructure to form an INVADER oligonucleotide for a second cleavagereaction. The structure formed between the secondary target, thesecondary probe and the uncut primary probe is depicted in FIG. 105, asthe right hand structure shown in step 2 a. This structure is recognizedand cleaved by the 5′ nucleases, albeit very inefficiently (i.e., atless than about 1% in most reaction conditions). Nonetheless, theresulting product is indistinguishable from the specific product, andthus may lead to a false positive result. The same effect can occur whenthe cleaved primary probe creates and integrated INVADER/target (IT)molecule, as described in Example 46; the formation of the undesirablecomplex is depicted schematically in FIG. 106, as the right handstructure shown in step 2 a.

The improvements provided by the inclusion of ARRESTOR oligonucleotidesof various compositions in each of these types of sequential INVADERassays are demonstrated in the following Examples. These ARRESTORoligonucleotides are configured to bind the residual uncut probe fromthe first cleavage reaction in the series, thereby increasing theefficacy of and reducing the non-specific background in the subsequentreaction(s).

Example 49 “ARRESTOR” Oligonucleotides Improve Sensitivity of MultipleSequential Invasive Cleavage Assays

In this Example, the effect of including an ARRESTOR oligonucleotide onthe generation of signal using the IT probe system depicted in FIGS. 97and 106 is demonstrated. The ARRESTOR oligonucleotide hybridizes to theprimary probe, mainly in the portion that recognizes the target nucleicacid during the first cleavage reaction. In addition to examining theeffects of adding an ARRESTOR oligonucleotide, the effects of usingARRESTOR oligonucleotides that extended in complementarity differentdistances into the region of the primary probe that composes thesecondary IT structure were also investigated. These effects werecompared in reactions that included the target DNA over a range ofconcentrations, or that lacked target DNA, in order to demonstrate thelevel of nonspecific (i.e., not related to target nucleic acid)background in each set of reaction conditions.

The target DNA for these reactions was a fragment that comprised thefull length of the hepatitis B genome from strain of serotype adw. Thismaterial was created using the polymerase chain reaction from plasmidpAM6 (ATCC #45020D). The PCRs were conducted using a vector-basedforward primer, oligo #156-022-001 (5′-ggcgaccacacccgtcctgt-3′; SEQ IDNO:168) and a reverse primer, oligo #156-022-02(5′-ccacgatgcgtccggcgtag-3′; SEQ ID NO:169) to amplify the full lengthof the HBV insert, an amplicon of about 3.2 kb. The cycling conditionsincluded a denaturation of the plasmid at 95° C. for 5 minutes, followedby 30 cycles of 95° C., 30 seconds; 60° C., 40 seconds; and 72° C., 4minutes. This was followed by a final extension at 72° C. for 10minutes. The resulting amplicon, termed pAM6#2, was adjusted to 2 MNH₄OAc, and collected by precipitation with isopropanol. After drying invacuo, DNA was dissolved in 10 mM Tris pH 0.0, 0.1 mM EDTA. Theconcentration was determined by OD₂₀₀ measurement, and by NVADER assaywith comparison to a standard of known concentration.

The INVADER reactions were conducted as follows. Five master mixes,termed “A,” “B,” “C,” “D,” and “E,” were assembled; all mixes contained12.5 mM MOPS, pH 7.5, 500 fmoles primary INVADER oligo #218-55-05 (SEQID NO:171), 10 ng human genomic DNA (Novagen) and 30 ng AfuFEN1 enzyme,for every 8 μl of mix. Mix A contained no added HBV genomic ampliconDNA; mix B contained 600 molecules of HBV genomic amplicon DNA pAM6 #2;mix C contained 6,000 molecules pAM6 #2; mix D contained 60,000molecules pAM6 #2; and mix E contained 600,000 molecules pAM6 #2. Themixes were aliquotted to the reaction tubes, 8 μl/tube: mix A to tubes1, 2, 11, 12, 21 and 22; mix B to tubes 3, 4, 13, 14, 23 and 24; mix Cto tubes 5, 6, 15, 16, 25 and 26; mix D to tubes 7, 8, 17, 18, 27 and28; and mix E to tubes 9, 10, 19, 20, 29 and 30. The samples wereincubated at 95° C. for 4 minutes to denature the HBV genomic ampliconDNA. The reactions were then cooled to 67° C., and 2 μl of a mixcontaining 37.5 mM MgCl₂ and 2.5 pmoles 218-95-06 (SEQ ID NO:183) forevery 2 μl, was added to each sample. The samples were incubated at 67°C. for 60 minutes. Three secondary reaction master mixes were prepared,all mixes contained 10 pmoles of secondary probe oligonucleotide#228-48-04 (SEQ ID NO:173) for every 2 μl of mix. Mix 2A contained noadditional oligonucleotide, mix 2B contained 5 pmoles “ARRESTOR” oligo#218-95-03 (SEQ ID NO:184) and mix 2C contained 5 pmoles of “ARRESTOR”oligo #218-95-01 (SEQ ID NO:174). After the 60 minute incubation at 67°C. (the primary reaction described above), 2 μl of the secondaryreaction mix was added to each sample: Mix 2A was added to samples#1-10; Mix 2B was added to samples #11-20; and Mix 2C was added tosamples #21-30. The temperature was adjusted to 52° C. and the sampleswere incubated for 30 minutes at 52° C. The reactions were then stoppedby the addition of 10 μl of a solution of 95% formamide, 5 mM EDTA and0.02% crystal violet. All samples were heated to 95° C. for 2 minutes,and 4 μl of each sample were resolved by electrophoresis through 20%denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffercontaining 45 mM Tris-Borate (pH8.3) and 1.4 mM EDTA. The results wereimaged using the Molecular Dynamics Fluoroimager 595, excitation 488,emission 530. The resulting images are shown in FIG. 107.

In FIG. 107, Panel A shows the results of the target titration when noARRESTOR oligonucleotide was included in the secondary reaction; Panel Bshows the results of the same target titration using an ARRESTORoligonucleotide that extended 2 nt into the non-target complementaryregion of the primary probe; and Panel C shows the results of the sametarget titration using an ARRESTOR oligonucleotide that extended 4 ntinto the non-target complementary region of the primary probe. Theproduct of the secondary cleavage reaction is seen as a band near thebottom of each panel. The first two lanes of each panel (i.e., 1 and 2,11 and 12, 21 and 22) lacked target DNA, and the signal the co-migrateswith the product band represents the nonspecific background under eachset of conditions.

It can be seen by visual inspection of these panels that the backgroundsignal is both reduced, and made more predictable, by the inclusion ofeither species of ARRESTOR oligonucleotides. In addition to reducing thebackground in the no-target control lanes, the background reduction inthe reactions that had the more dilute amounts of target included isreduced, leading to a signal that is a more accurate reflection of thetarget contained within the reaction, thus improving the quantitativerange of the multiple, sequential invasive cleavage reaction.

To quantify the impact of including the ARRESTOR oligonucleotide in thesecondary cleavage reaction under these conditions, the average productband signal from the reactions having the largest amount of target(i.e., averages of the signals from lanes 9 and 10, lanes 19 and 20, andlanes 29 and 30), were compared to the averaged signal from theno-target contol lanes for each panel, determine the “fold overbackground,” the factor of signal amplification over background, undereach set of conditions. For the reactions without the ARRESTORoligonucleotide, Panel A, the fold over background was 5.3; for Panel B,the fold over background was 12.7; and for Panel C, the fold overbackground was 13.4, indicating that in this system inclusion of anyARRESTOR oligonucleotide at least doubled the specificity of the signalover the ARRESTOR oligonucleotide -less reactions, and that the ARRESTORoligonucleotide that extended slightly farther into the non-targetcomplementary region may be slightly more effective, at least in thisembodiment of the system. This clearly shows the benefits of using anARRESTOR oligonucleotide to enhance the specificity of these reactions,an advantage that is of particular benefit at low levels of targetnucleic acid.

Example 50 “ARRESTOR” Oligonucleotides Allow Use of HigherConcentrations of Primary Probe Without Increasing Background Signal

It was demonstrated in Example 36, that increasing the concentration ofthe probe in the invasive cleavage reaction could dramatically increasethe amount of signal generated for a given amount of target DNA. Whilenot intending to limit the explanation to any specific mechanism, thisis believed to be caused by the fact that increased concentration ofprobe increases the rate at which the cleaved probe is supplanted by anuncleaved copy, thereby increasing the apparent turnover rate of thecleavage reaction. Unfortunately, this effect could not heretofore beapplied in the primary cleavage reaction of a multiple sequentialINVADER assay because the residual uncleaved primary probe can hybridizeto the secondary target, in competition with the cleaved molecules,thereby reducing the efficacy of the secondary reaction. Elevatedconcentrations of primary probe exacerbate this problem. Further, theresulting complexes, as described above, can be cleaved at a low level,contributing to background. Therefore, increasing the primary probe canhave the double negative effect of both slowing the secondary reactionand increasing the level of this form of non target-specific background.The use of an ARRESTOR oligonucleotide to sequester or neutralize theresidual primary probe allows this concentration-enhancing effect to beapplied to these sequential reactions.

To demonstrate this effect, two sets of reactions were conducted. In thefirst set of reactions, the reactions were conducted using a range ofprimary probe concentrations, but no ARRESTOR oligonucleotide wassupplied in the secondary reaction. In the second set of reactions, thesame probe concentrations were used, but an ARRESTOR oligonucleotide wasadded for the secondary reactions.

All reactions were performed in duplicate. Primary INVADER reactionswere done in a final volume of 10 μl and contained: 10 mM MOPS, pH 7.5,7.5 mM MgCl₂, 500 fm of primary INVADER (218-55-05; SEQ ID NO:171); 30ng of AfuFEN1 enzyme and 10 ng of human genomic DNA. 100 zeptomoles ofHBV pAM6 #2 amplicon was included in all even numbered reactions (byreference to FIGS. 108A and B). Reactions included 10 pmoles, 20 pmoles,50 pmoles, 100 pmoles or 150 pmoles of primary probe (218-55-02; SEQ IDNO:170). MOPS, target and INVADER oligonucleotides were combined to afinal volume of 7 μl. Samples were heat denatured at 95° C. for 5minutes, then cooled to 67° C. During the 5 minute denaturation, MgCl₂,probe and enzyme were combined. The primary INVADER reactions wereinitiated by the addition of 3 μl of MgCl₂, probe and enzyme mix, to thefinal concentrations indicated above. Reactions were incubated for 30minutes at 67° C. The reactions were then cooled to 52° C., and eachprimary INVADER reaction received the following secondary reactioncomponents in a total volume of 4 μl: 2.5 pmoles secondary target (oligonumber 218-95-04; SEQ ID NO:172); 10 pmoles secondary probe (oligonumber 228-48-04; SEQ ID NO:173). The reactions that included theARRESTOR oligonucleotide had either 40 pmoles, 80 pmoles, 200 pmoles,400 pmoles or 600 pmoles of ARRESTOR oligonucleotide (oligo number218-95-01; SEQ ID NO:174), added at a 4-fold molar excess over theprimary probe amount for each reaction, with this mix. Reactions werethen incubated at 52° C. for 30 minutes. The reactions were stopped bythe addition of 10 μl of a solution of 95% formamide, 10 mM EDTA and0.02% crystal violet. All samples were heated to 95° C. for 1 minute,and 4 μl of each sample were resolved by electrophoresis through 20%denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffercontaining 45 mM Tris-Borate (pH_(8.3)) and 1.4 mM EDTA. The resultswere imaged using the Molecular Dynamics Fluoroimager 595, excitation488, emission 530. The resulting images for the reactions either withoutor with an ARRESTOR oligonucleotide are shown in FIGS. 108A and 108B,respectively. The products of cleavage of the secondary probe are seenas a band near the bottom of each panel.

In FIG. 108A, lane sets 1 and 2 show results with 10 pmoles of primaryprobe; 3 and 4 had 20 pmoles; 5 and 6 had 50 pmoles; 7 and 8 had 100pmoles; and 9 and 10 had 150 pmoles. It can be seen by visualexamination, that the increases in the amount of primary probe have thecombined effect of slightly increasing the background in the no-targetlanes (odd numbers) while reducing the specific signal in the presenceof target (even numbered lanes), and therefore the reducing thespecificity of the reaction if viewed as the measure of “fold overbackground,” demonstrating that the approach of increasing signal byincreasing probe cannot be applied in these sequential reactions.

In FIG. 108B, lane sets 1 and 2 show results with 10 pmoles of primaryprobe; while 3 and 4 had 20 pmoles; 5 and 6 had 50 pmoles; 7 and 8 had100 pmoles; and 9 and 10 had 150 pmoles. In addition, each reactionincluded 4-fold molar excess of the ARRESTOR oligonucleotide addedbefore the secondary cleavage reaction. It can be seen by visualexamination that the background in the no-target lanes (odd numbers) islower in all cases, while the specific signal in the presence of target(even numbered lanes) increases with increased amounts of primary probe,leading to a greater “fold over background” sensitivity at this targetlevel.

To quantitatively compare these effects, the fluorescence signal fromthe products of both non-specific and specific cleavage were measured.The results are depicted graphically in FIG. 108C, graphed as a measureof the percentage of the secondary probe cleaved during the reaction,compared to the amount of primary probe used. Examination of the plotsfrom the no-target reactions confirms that the background in the absenceof the ARRESTOR oligonucleotide is, in general, roughly two-fold higher,and that both increase slightly with the increasing probe amounts. Thespecific signals however, diverge between the two sets of reaction moredramatically. While the signal in the no-ARRESTOR oligonucleotidereactions decreases steadily as primary probe was increased, the signalin the ARRESTOR oligonucleotide reactions continued to increase. At thehighest primary probe concentrations tested, the no-ARRESTORoligonucleotide reactions had specific signal that was only 1.7 foldover background, while the ARRESTOR oligonucleotide reactions detectedthe 100 zmoles (60,000 copies) of target with a signal 6.5 fold overbackground, thus demonstrating the improvement in the sequentialinvasive cleavage reaction when an ARRESTOR oligonucleotide is included.

Example 51 Modified Backbones Improve Performance of ARRESTOROligonucleotides All Natural “ARRESTOR” Oligo With No 3′-Amine

The reactions described in the previous two Examples used ARRESTORoligonucleotides that were constructed using 2′ O-methyl ribosebackbone, and that included a positively charged amine group on the 3′terminal nucleotide. The modifications were made specifically to reduceenzyme interaction with the primary probe/ARRESTOR oligonucleotidecomplex. During the development of the present invention, it wasdetermined that the 2′ O-methy modified oligonucleotides are somewhatresistant to cleavage by the 5′ nucleases, just as they are slowlydegraded by nucleases when used in antisense applications (See e.g.,Kawasaki et al., J. Med. Chem., 36:831 [1993]).

Further, as demonstrated in Example 35, the presence of an amino groupon the 3′ end of an oligonucleotide reduces its ability to directinvasive cleavage. To reduce the possibility that the ARRESTORoligonucleotide would form a cleavage structure in this way, an aminogroup was included in the design of the experiments described in thisand other Examples.

Initial designs of the ARRESTOR oligonucleotides (sometimes referred toas “blockers”) did not include these modifications, and these moleculeswere found to provide no benefit in reducing background cleavage in thesequential invasive cleavage assay and, in fact, sometimes contributedto background by inducing cleavage at an unanticipated site, presumablyby providing some element to an alternative cleavage structure. Theeffects of natural and modified ARRESTOR oligonucleotide on thebackground noise in these reactions are examined in this Example.

The efficacy of an “all-natural ARRESTOR oligonucleotide (i.e., anARRESTOR oligonucleotide that did not contain any base analogs ormodifications) was examined by comparison to an identical reactions thatlacked ARRESTOR oligonucleotide. All reactions were performed induplicate, and were conducted as follows. Two master mixes wereassembled, each containing 12.5 mM MOPS, pH 7.5, 500 fmoles primaryINVADER oligonucleotide #218-55-05 (SEQ ID NO:171), 10 ng human genomicDNA (Novagen) and 30 ng AfuFEN1 enzyme for every 8 μl of mix. Mix Acontained no added HBV genomic amplicon DNA, mix B contained 600,000molecules of HBV genomic amplicon DNA, pAM6 #2. The mixes weredistributed to the reaction tubes, in aliquots of 8 μl/tube as follows:mix A to tubes 1, 2, 5 and 6; and mix B to tubes 3, 4, 7 and 8. Thesamples were incubated at 95° C. for 4 minutes to denature the HBVgenomic amplicon DNA. The reactions were then cooled to 67° C. and 2 ulof a mix containing 37.5 mM MgCl₂ and 10 pmoles 218-55-02B (SEQ IDNO:185) for every 2 μl, was added to each sample. The samples were thenincubated at 67° C. for 30 minutes. Two secondary reaction master mixeswere prepared, each containing 10 pmoles of secondary probe oligo#228-48-04N (SEQ ID NO:178) and 2.5 pmoles of secondary targetoligonucleotide #218-95-04 (SEQ ID NO:172) for every 3 μl of mix. Mix 2Acontained no additional oligonucleotide, while mix 2B contained 50pmoles of the natural “ARRESTOR” oligonucleotide #241-62-02 (SEQ IDNO:186). After the initial 30 minute incubation at 67° C., thetemperature was adjusted to 52° C., and 3 μl of a secondary reaction mixwas added to each sample, as follows: Mix 2A was added to samples #1-4;and Mix 2B was added to samples #5-8. The samples were then incubatedfor 30 minutes at 52° C. The reactions were then stopped by the additionof 10 μl of a solution of 95% formamide, 10 mM EDTA and 0.02% crystalviolet.

All of the samples were heated to 95° C. for 2 minutes, and 4 μl of eachsample were resolved by electrophoresis through a 20% denaturingacrylamide gel (19: 1 cross-linked) with 7 M urea, in a buffercontaining 45 mM Tris-Borate (pH8.3) and 1.4 mM EDTA. The results wereimaged using the Molecular Dynamics Fluoroimager 595, excitation 488,emission 530. The resulting image is shown in FIG. 109A.

To compare the effects of the various modifications made to the ARRESTORoligonucleotides, reactions were performed using ARRESTORoligonucleotides having all natural bases, but including a 3′ terminalamine; ARRESTOR oligonucleotide having the 3′ portion composed of 2′O-methyl nucleotides, plus the 3′ terminal amine; and ARRESTORoligonucleotide composed entirely of 2′ O-methyl nucleotides, plus the3′ terminal amine. These were compared to reactions performed without anARRESTOR oligonucleotide. The reactions were conducted as follows. Twomaster mixes were assembled, all mixes contained 14.3 mM MOPS, pH 7.5,500 fmoles primary INVADER oligo #218-55-05 (SEQ ID NO:171) and 10 nghuman genomic DNA (Novagen) for every 7 μl of mix. Mix A contained noadded HBV genomic amplicon DNA, mix B contained 600,000 molecules of HBVgenomic amplicon DNA, pAM6 #2. The mixes were distributed to thereaction tubes, at 7 μl/tube: mix A to tubes 1, 2, 5, 6, 9, 10, 13 and14; and mix B to tubes 3, 4, 7, 8, 11, 12, 15 and 16. The samples werewarmed to 95° C. for 4 minutes to denature the HBV DNA. The reactionswere then cooled to 67° C. and 3 μl of a mix containing 25 mM MgCl₂, 25pmoles 218-55-02B (SEQ ID NO:185) and 30 ng AfuFEN1 enzyme per 3 μl,were added to each sample. The samples were then incubated at 67° C. for30 minutes. Four secondary reaction master mixes were prepared; allmixes contained 10 pmoles of secondary probe oligonucleotide #228-48-04B(SEQ ID NO:190) and 2.5 pmoles of secondary target oligonucleotide#218-95-04 (SEQ ID NO:172) for every 3 μl of mix. Mix 2A contained noadditional oligonucleotide, while mix 2B contained 100 pmoles of thenatural+amine ARRESTOR oligonucleotide #241-62-01 (SEQ ID NO:187), mix2C contained 100 pmoles of partially O-methyl+amine oligonucleotide#241-62-03 (SEQ ID NO:188) and mix 2D contained 100 pmoles of allO-methyl+amine oligonucleotide #241-64-01 (SEQ ID NO:189). After theinitial 30 minute incubation at 67° C., the temperature was adjusted to52° C. and 3 μl of a secondary reaction mix was added to each sample, asfollows: mix 2A was added to samples #1-4; mix 2B was added to samples#5-8; mix 2C was added to samples #9-12; and mix 2D was added to samples#13-16. The samples were incubated for 30 minutes at 52° C., thenstopped by the addition of 10 μl of a solution of 95% formamide, 10 mMNaEDTA, and 0.2% crystal violet.

All samples were heated to 95° C. for 2 minutes, and 4 μl of each samplewere resolved by electrophoresis through a 20% denaturing acrylamide gel(19:1 cross-linked) with 7 M urea, in a buffer containing 45 mMTris-Borate (pH8.3) and 1.4 mM EDTA. The results were imaged using theMolecular Dynamics Fluoroimager 595, excitation 488, emission 530. Theresulting image is shown in FIG. 109B.

In FIG. 109A, the left hand panel shows the reactions that lacked anARRESTOR oligonucleotide, while the right hand panel shows the data fromreactions that included the all natural ARRESTOR oligonucleotide. Thefirst two lanes of each panel are from no-target controls, the secondset of lanes contained target. The products of cleavage are visible inthe bottom one/fourth of each panel. The position at which the specificreaction products should run is indicated by arrows on left and right.

It can be seen by examination of these data, that the reactions run inthe absence of ARRESTOR oligonucleotide show reproducible qualitybetween the replicates, and show significant cleavage only when targetis present. In contrast, the addition of another unmodifiedoligonucleotide into the reactions causes great variation between thereplicate lanes (e.g., lanes 5 and 6 were provided with the samereactants, but produced markedly different results). The introduction ofthe all natural ARRESTOR oligonucleotide produced, rather than reduced,background in these no-target lanes, and increased cleavage at othersites (i.e., the bands other that those indicated by the arrows flankingthe panels). For these reasons the modifications that are describedabove, the effects of which are shown on FIG. 109B, were incorporated.

The first 4 lanes of FIG. 109B show the products of duplicate reactionswithout an ARRESTOR oligonucleotide, plus or minus the HBV target (lanes1, 2, and lanes 3, 4, respectively); The next 4 lanes, 5, 6 and 7, 8used a natural ARRESTOR oligonucleotide having a 3′ terminal amine;lanes 9, 10 and 11, 12 used the ARRESTOR oligonucleotide with a 3′portion composed of 2′ O-methyl nucleotides, and having a 3′ terminalamine; lanes 13, 14 and 15, 16 used the ARRESTOR oligonucleotidecomposed entirely of 2′ O-methyl nucleotides and having a 3′ terminalamine. The products of cleavage of the secondary probe are visible inthe lower one third of each panel.

Visual inspection of these data shows that the addition of the 3′terminal amine to the natural ARRESTOR oligonucleotide suppresses theaberrant cleavage seen in FIG. 109A, but this ARRESTOR oligonucleotidedoes not improve the performance of the reaction, as compared to theno-ARRESTOR oligonucleotide controls. In contrast, the use of the 2′O-methyl nucleotides in the body of the ARRESTOR oligonucleotide doesreduce background, whether partially or completely substituted. Toquantify the relative effects of these modifications, the fluorescencefrom each of the co-migrating product bands was measured, the signalsfrom the duplicate lanes were averaged and the “fold over background”was calculated for each reaction containing target nucleic acid.

When ARRESTOR oligonucleotide was omitted, the target specific signal(lanes 3, 4) was 27-fold over the no target background; the naturalARRESTOR oligonucleotide +amine gave a signal of 17-fold overbackground; the partial 2′ O-methyl+amine gave a signal of 47-fold overbackground; and the completely 2′ O-methyl+amine gave a signal of 33fold over background.

These Figures show that both modifications can have a beneficial effecton the specificity of the multiple, sequential invasive cleavage assay.They also show that the use of the 2′ O-methyl substituted backbone,either partial or entire, markedly improves the specificity of thesereactions. It is intended that in various embodiments of the presentinvention, that any number of modifications that make either theARRESTOR oligonucleotide or the complex it forms with the primary targetresistant to nucleases will provide similar enhancement.

Example 52 Effect of ARRESTOR Oligonucleotide Length on SignalEnhancement in Multiple Sequential Invasive Cleavage Assays

As noted in the Description of the Invention, the optimal length for anARRESTOR oligonucleotide depends upon the design of the other nucleicacid elements of the INVADER reaction, particularly on the design of theprimary probe. In this Example, the effects of varying the length of theARRESTOR oligonucleotide were explored in systems using two differentsecondary probes. A schematic diagram showing these ARRESTORoligonucleotides aligned as they would hybridize to the primary probeoligonucleotide is provided in FIG. 110C. In this Figure, the region ofthe primary probe that recognizes the target nucleic acid is shownunderlined; the non-underlined portion, plus the first underlined baseis the portion that is released by the first cleavage, and goes on toparticipate in the second or subsequent cleavage structure.

All reactions were performed in duplicate. The INVADER reactions weredone in a final volume of 10 μl final volume containing 10 mM MOPS, pH7.5, mM MgCl₂, 500 fmoles of primary INVADER 241-95-01, (SEQ ID NO:176),25 pmoles of primary probe 241-95-02 (SEQ ID NO:175), 30 ng of AfuFEN1enzyme, and 10 ng of human genomic DNA, and if included, 1 amoles of HBVamplicon pAM 6 #2. MOPS, target DNA, and INVADER oligonucleotides werecombined to a final volume of 7 μl. Samples were heat denatured at 95°C. for 5 minutes, then cooled to 67° C. During the 5 minutedenaturation, MgCl₂, probe and enzyme were combined. The primary INVADERreactions were initiated by the addition of 3 μl of MgCl₂, probe andenzyme mix, to the final concentrations indicated above. Reactions wereincubated for 30 minutes at 67° C. The reaction were then cooled to 52°C., and each primary INVADER reaction received the following secondaryreaction components in a total volume of 3 μl: 2.5 pmoles secondarytarget 241-95-07 (SEQ ID NO:177), 10 pmoles of either secondary probe228-48-04 (SEQ ID NO:173), or 228-48-04N (SEQ ID NO:178) and 100 pmolesof an ARRESTOR oligonucleotide, either 241-95-03 (SEQ ID NO:179),241-95-04 (SEQ ID NO:180), 241-95-05 (SEQ ID NO:181) or 241-95-06 (SEQID NO:182). The ARRESTOR oligonucleotide were omitted from somereactions as controls for ARRESTOR oligonucleotide effects.

The reactions were incubated at 52° C. for 34 minutes, and were thenstopped by the addition of 10 μl of 95% formamide, 10 mM EDTA, and 0.02%crystal violet. All samples were heated to 95° C. for 1 minute, and 4 μlof each sample were resolved by electrophoresis through 20% denaturingacrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer containing45 mM Tris-Borate (pH8.3) and 1.4 mM EDTA. The results were imaged usingthe Molecular Dynamics Fluoroimager 595, excitation 488, emission 530.The resulting images for the reactions with the shorter and longersecondary probes are shown in FIGS. 110A and 110B, respectively.

In each Figure, the products of cleavage are visible as bands in thebottom half of each lane. The first 4 lanes of each Figure show theproducts of duplicate reactions without an ARRESTOR oligonucleotide,plus or minus the HBV target (lanes sets 1 and 2 respectively); in thenext 4 lanes, sets 3 and 4 used the shortest ARRESTOR 241-95-03 (SEQ IDNO:179); lanes 5 and 6 used 241-95-04 (SEQ ID NO:180); lanes 7 and 8used 241-95-05 (SEQ ID NO:181); and lanes 9 and 10 used 241-95-06 (SEQID NO:182).

The principal background of concern is the band that appears in the “notarget” control lanes (odd numbers; this band co-migrates with thetarget-specific signal near the bottom of each gel panel). Visualinspection shows that the shortest ARRESTOR oligonucleotide was theleast effective at suppressing this background, and that the efficacywas increased when the ARRESTOR oligonucleotide extended further intothe portion that participates in the subsequent cleavage reaction. Evenwith this difference in effect, it can be seen from these data thatthere is much latitude in the design of the ARRESTOR oligonucleotide.The choice of lengths will be influenced by the temperature at which thereaction making use of the ARRESTOR oligonucleotide is performed, thelengths of the duplexes formed between the primary probe and the target,the primary probe and the secondary target, and the relativeconcentrations of the different nucleic acid species in the reactions.

Example 53 Effect of ARRESTOR Oligonucleotide Concentration on SignalEnhancement in Multiple Sequential Invasive Cleavage Assays

In examining the effects of including ARRESTOR oligonucleotides in thesecleavage reactions, it was of interest to determine if the concentrationof the ARRESTOR oligonucleotide in excess of the primary probeconcentration would have an effect on yields of either non-specific orspecific signal, and if the length of the ARRESTOR oligonucleotide wouldbe a factor. These two variable were investigated in the followingExample.

All reactions were performed in duplicate. The primary INWADER reactionswere done in a final volume of 10 μl and contained 10 mM MOPS, pH 7.5;7.5 mM MgCl₂, 500 fmoles of primary INVADER 241-95-01 (SEQ ID NO:176),25 pmoles of primary probe 241-95-02 (SEQ ID NO:175), 30 ng of AfuFEN1enzyme, and 10 ng of human genomic DNA. Where included, the target DNAwas 1 amole of HBV amplicon pAM 6 #2, as described above. MOPS, targetand INVADER were combined to a final volume of 7 μl. The samples wereheat denatured at 95° C. for 5 minutes, then cooled to 67° C. During the5 minute denaturation, MgCl₂, probe and enzyme were combined. Theprimary INVADER reactions were initiated by the addition of 3 μl ofMgCl₂, probe and enzyme mix. The reactions were incubated for 30 minutesat 67° C. The reactions were then cooled to 52° C. and each primaryINVADER reaction received the following secondary reaction components:2.5 pmoles secondary target 241-95-07 (SEQ ID NO:177), 10 pmolessecondary probe 228-48-04 (SEQ ID NO:173); and, if included, 50, 100 or200 pmoles of either ARRESTOR oligonucleotide 241-95-03 (SEQ ID NO:179)or 241-95-05 (SEQ ID NO:181), in a total volume of 3 μl. Reactions werethen incubated at 52° C. for 35 minutes. Reactions were stopped by theaddition of 10 μl of 95% formamide, 10 mM EDTA, and 0.02% crystalviolet. All of the samples were heated to 95° C. for 1 minute, and 4 μlof each sample were resolved by electrophoresis through 20% denaturingacrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer containing45 mM Tris-Borate (pH8.3), and 1.4 mM EDTA. The results were imagedusing the Molecular Dynamics Fluoroimager 595, excitation 488, emission530. The resulting images are shown as a composite image in FIG. 111.

Each of the duplicate reactions were loaded on the gel in adjacent lanesand are labeled with a single lane number. All odd numbered lanes wereno-target controls. Lanes 1 and 2 had no ARRESTOR oligonucleotide added;lanes 3-8 show results from reactions containing the shorter ARRESTORoligonucleotide, 241-95-03 (SEQ ID NO:179); lanes 9-14 show results fromreactions containing the longer ARRESTOR oligonucleotide, 241-95-05 (SEQID NO:181). The products of cleavage from the secondary reaction arevisible in the bottom one third of each panel. Visual inspection ofthese data (i.e., comparison of the specific products to the backgroundbands) shows that both ARRESTOR oligonucleotides have some beneficialeffect at all concentration.

To quantify the relative effects of ARRESTOR oligonucleotide length andconcentration, the fluorescence from each of the co-migrating productbands was measured, the signals from the duplicate lanes were averagedand the “fold over background” (signal+target/signal-target) wascalculated for each reaction containing target nucleic acid. Thereaction lacking an ARRESTOR oligonucleotide yielded a signalapproximately 27-fold over background. Inclusion of the shorter ARRESTORoligonucleotide at 50, 100 or 200 pmoles produced products at 42, 51 and60-fold over background, respectively. This shows that while the shortarrestr at the lowest concentration seems to be less effective than thelonger ARRESTOR oligonucleotides (See, previous Example) this can becompensated for by increasing the concentration of ARRESTORoligonucleotide, and thereby the ARRESTOR oligonucleotide:primary proberatio.

In contrast, inclusion of the longer ARRESTOR oligonucleotide at 50, 100or 200 pmoles produced products at 60, 32 and 24 fold over background,respectively. At the lowest concentration, the efficacy of this longerARRESTOR oligonucleotide relative to the shorter ARRESTORoligonucleotide is consistent with the previous Example. Increasing theconcentration, however, decreased the yield of specific product,suggesting a competition effect with some element of the secondarycleavage reaction.

These data show that the ARRESTOR oligonucleotides can be used toadvantage in a number of specific reaction designs. The choice ofconcentration will be influenced by the temperature at which thereaction making use of the ARRESTOR oligonucleotide is performed, thelengths of the duplexes formed between the primary probe and the target,the primary probe and the secondary target, and between the primaryprobe and the ARRESTOR oligonucleotide.

Selection of oligonucleotides for target nucleic acids other than theHBV shown here, (e.g., oligonucleotide composition and length), and theoptimization of cleavage reaction conditions in accord with the modelsprovided here follow routine methods and common practice well known tothose skilled in the methods of molecular biology.

Example 45 demonstrated that some enzymes require an overlap between anupstream INVADER oligonucleotide and a downstream probe oligonucleotideto create a cleavage structure (FIG. 100). It has also been determinedthat the 3′ terminal nucleotide of the INVADER oligonucleotide need notbe complementary to the target strand, even if it is the onlyoverlapping base in the INVADER oligonucleotide (e.g., as with the HCMVprobes shown in FIG. 103). The requirement for an overlap can serve as aconvenient basis for detecting single base polymorphisms (SNPs) ormutations in a nucleic acid sample.

For detection of single base variations, at least two oligonucleotides(e.g., a probe and an INVADER oligonucleotide) hybridize in tandem tothe target nucleic acid to form the overlapping structure recognized bythe CLEAVASE enzyme to be used in the reaction. An unpaired “flap” isincluded on the 5′ end of the probe. The enzyme recognizes the overlapand cleaves off the unpaired flap, releasing it as a target-specificproduct. Enzymes that have a strong preference for an overlappingstructure, i.e., that cleave the overlapping structure at a much greaterrate than they cleave a non-overlapping structure include the FEN-1enzymes from Archaeoglobus fulgidus and Pyrococcus furiosus and suchenzymes are particularly preferred in the detection of mutations andSNPs. In the secondary reaction, the released flap serves as an INVADERoligonucleotide to create another overlapping cleavage structure (e.g.,as shown in FIG. 96). In the following examples, the released flapcreates this overlapping structure in conjunction with a FRET cassette,a single oligonucleotide having a region of self-complementarity to forma hairpin (FIG. 112A) When the FRET cassette is cleaved to release its5′ nucleotide, the fluorescent dye (F) and the quencher (Q) on thecassette are separated and a detectable fluorescence signal is produced.If the probe and the target sequence do not match perfectly at thecleavage site (e.g., as in FIG. 112B), the overlapped structure does notform, cleavage is suppressed, and no fluorescence will be produced.

The reactions may be performed under conditions in which the probes andFRET cassettes turn over continuously without temperature cycling orcleavage. When an uncut probe hybridizes to the target next to anoverlapping INVADER oligonucleotide, the probe can be cleaved to producea target-specific product, which in turn enables the cleavage of manyFRET cassettes.

The following eight examples demonstrate the design and application ofthe sequential INVADER assay with FRET cassette detection to analysis ofSNPs and mutations in a variety of nucleic acid samples.

Example 54 Detection of Single Nucleotide Polymorphisms in the HumanApolipoprotein E Gene

This Example describes an assay for the detection of SNPs in the humanApolipoprotein E gene. Probe and INVADER oligonucleotides were designedto target single nucleotide polymorphisms (SNPs) at two positions in thehuman apolipoprotein E (apoE) gene. Three different alleles exist forthe apoE gene, epsilon 2, epsilon 3, and epsilon 4, which code for 3different isoforms of the apoE protein, termed E2, E3 and E4. Thedifferent isoforms vary in at amino acid positions 112 or 158 (see TableA), and each variation is caused by a single base change in thecorresponding codon.

TABLE A 112 158 ISOFORM codon amino acid codon amino acid E2 TGC cys TGCcys E3 TGC cys CGC arg E4 CGC arg CGC arg

INVADER and probe oligonucleotides were designed using the algorithmdescribed above (detailed description of the invention), with the probeset selected to operate at 63° C. As shown in FIGS. 113A-D, one INVADERoligonucleotide and two unlabelled, probe oligonucleotides, one for eachvariant at a given locus, were designed for each codon. For codon 112,the probes were designed to detect either the C nucleotide (to code forarginine; SEQ ID NO:197) or a T nucleotide (to code for cysteine; SEQ IDNO:198). For codon 158, the probes were designed to detect either a Cnucleotide (to code for arginine; SEQ ID NO:199) or a T nucleotide (tocode for cysteine; SEQ ID NO:200). A FRET cassette to be used with allof the probe sets was also synthesized (SEQ ID NO:201, FIG. 113). Inthis Example and in the following Examples, all oligonucleotides weresynthesized using standard phosphoramidite chemistries. Primary probeoligonucleotides were unlabeled. The FRET cassettes were labeled by theincorporation of Cy3 phosphoramidite and fluorescein phosphoramidite(Glen Research). While designed for 5′ terminal use, the Cy3phosphoramidite has an additional monomethoxy trityl (MMT) group on thedye that can be removed to allow further synthetic chain extension,resulting in an internal label with the dye bridging a gap in thesugar-phosphate backbone of the oligonucleotide (as diagrammed in thepanels of FIG. 113). While a nucleotide may be omitted at this positionto accommodate the dye, we have determined it is not necessary, and nonucleotides were omitted from the FRET cassettes used in these examples.Amine or phosphate modifications, where indicated, were used on the 3′ends of the primary probes and the FRET cassettes to prevent their useas invasive oligonucleotides. 2′-O-methyl bases in the secondary targetoligonucleotides are indicated by underlining and were also used tominimize enzyme recognition of 3′ ends. In addition, reactions havingsynthetic target DNAs were used as positive controls to verify theactivity of the reaction components. The control targets are illustratedin FIGS. 113A-D. The 112 arg, 112 cys, 158 arg, and 158 cys controloligonucleotides are SEQ ID NOS:191 -194, respectively.

Genomic DNA sample AG09714 was purchased from Coriell. This sample wasquantitated via Pico Green and diluted with Tris with 0.1 mM EDTA to aconcentration of approximately 100 ng/μl. This sample (9714 in FIG.114A) was used to test for the 112 mutation. Samples 39634, 32435 and31071 were purchased as whole blood from Lampire Biological Labs., Inc.Samples 511, 537, 538 and 539 were whole blood samples donated by theBlood Center of Southeast Wisconsin (Milwaukee, Wis.). Genomic DNA wasprepared via the PUREGENE Blood Kit (Gentra) according to themanufacturer's instructions. Samples 39634 and 32435 were used to testfor the 112 mutation, and sample 31071, 511, 537, 538 and 539 were usedto test for the 158 mutation. One μl of genomic DNA was used perreaction, with 9 μl of water, for a final volume of 10 μl. Althoughdetermination of the full genotype for any one sample generally requiresanalysis of both loci, the samples listed above were selected to showrepresentative signals from, and thus the functioning of, each probeset. Complete genotyping requires each sample to be tested with bothprobe sets.

The experiment comprised testing each genomic DNA for the indicatedalleles, along with reactions having no target DNA to allow measurementof any background signal not attributable to the presence of a targetsequence. Reactions testing the 112 locus were done in quadruplicate,reactions testing the 158 locus were done in triplicate.

Reaction components were prepared as batch mixes for dispensing to theindividual test reactions. Batches of INVADER mix for each allele wereprepared, comprising for each planned reaction: 4 μl of 16% PEG 8000/50mM MOPS pH 7.5 and 1 μl of 1 μM INVADER oligonucleotide (either the 112or the 158). Batches of CLEAVASE enzyme/Mg²⁺/probe mix for each allelewere prepared, comprising for each planned reaction: 2 μl of 75 mMMgCl₂, 1 μl of 10 μM FRET cassette, 1 μl 10 μM probe oligonucleotide(any one of the 112 or the 158 T or C probe oligonucleotides), and 1 μlof 200 ng/μl Afu FEN-1 enzyme.

For each reaction, 5 μl of INVADER reaction mix were aliquoted into eachwell of a 96-well Low Profile Polypropylene Microplate (MJ Research,).Ten μl of each control DNA or genomic sample (approximately 100 ng-190ng) were added and mixed by pipetting up and down. The no-targetcontrols received 1 μg of yeast tRNA instead of target DNA. Thereactions comprising the synthetic targets as positive controls includedeither 150 or 100 zeptomoles (zmoles) of the 112 or 158 synthetictargets, respectively, and 1 μg of yeast tRNA. Each reaction wasoverlaid with 20 μl of clear CHILLOUT liquid wax, and incubated at 95°C. for 5 minutes. The reaction temperature was then lowered to 63° C., 5μl of the appropriate CLEAVASE enzyme/Mg²⁺/Probe reaction mix was addedto each reaction and mixed by pipetting up and down 3-5 times, and thereactions were further incubated for 4 hours at 63° C., and were readdirectly on a CYTOFLUOR Series 4000 Fluorescence Multi-well PlateReader, (PerSeptive Biosystems), using the following settings:Excitation (wavelength/bandwidth): 485/20 nm; Emission(wavelength/bandwidth): 530/25 nm; Gain: 37. The net averagedfluorescence signal was calculated by subtracting the averaged no-targetsignal (background) from the corresponding averaged target DNA reactionsignal and the data were plotted using Excel spreadsheet software(Microsoft).

Results for the ApoE 112 and ApoE 158 loci are shown graphically inFIGS. 114A and 114B, respectively, with target DNAs indicated on thehorizontal axis and the fluorescence units indicated on the verticalaxis. The white bars represent the net averaged signal from the “C”probe, while the dark bars represent the net averaged signal from the“T” probe. The reactions having synthetic targets are indicated as Syn Cand Syn T for the C and T controls, respectively. At both loci, thesamples that are homozygous for either the C or T allele are easilyidentified by having a strong signal from only one probe or the other,while the heterozygous samples, are easily identified by having strongsignals from both the C and T probes.

Example 55 Detection of Mutations in the Human Hemochromatosis (HFE)Gene

The human hemochromatosis (HFE gene) gene is located in the MHC regionof chromosome 6, and was initially named HLA-H. It was later renamed theHFE gene, in accordance with the WHO Nomenclature Committee for Factorsof the HLA System. Two different, single-base variations in the HFE geneare responsible for the vast majority of the cases of hereditaryhemochromatosis, or iron overload disease. The most common variant,termed C282Y, is caused by a change from the wild type (WT) adenine (A)to the mutant (MT) guanine (G) at codon 282 that causes an amino acidchange from a cysteine to a tyrosine residue. The second variationcommonly detected in individuals suffering from iron overload disorderis termed H63D, and is caused by a WT cytosine (C) to a MT guanine (G)change at codon 63 that causes an amino acid change from a histidine toan aspartic acid residue in the expressed protein.

INVADER and probe oligonucleotide sets were designed as described aboveto detect both the WT and MT alleles for both the C282Y and H63D sitesand are shown in FIGS. 115A-D. Detection of the C282 polymorphism usedone INVADER oligonucleotide (SEQ ID NO:202), one probe specific for eachvariant of the allele (WT and MT, SEQ ID NOS:208 and 209, respectively)and a first FRET cassette (SEQ ID NO:210). Oligonucleotides for the H63locus included an INVADER oligonucleotide (SEQ ID NO:203), one probespecific for each variant of the allele (WT and MT, SEQ ID NOS:211 and212, respectively), and a second FRET cassette (SEQ ID NO:213). Inaddition, reactions having synthetic target DNAs were used as positivecontrols to verify the activity of the reaction components. The controltargets are illustrated in FIGS. 115 A-D. The C282 WT, C282 MT, H63 WTand H63 MT control oligonucleotides are SEQ ID NOS: 204 -207,respectively. Human genomic DNA samples 14640, 14641, 14646, 14690, and14691 were purchased from Coriell Cell Repositories (Catalog #s NA14640,NA14641, NA1446, NA14690, and NA14691).

Reactions were performed and analyzed as described in Example 54. Thereactions comprising the synthetic targets as positive controls included100 zmoles of the synthetic target and 1 μg of yeast tRNA.

Results are shown graphically in FIG. 116, with target DNAs indicated onthe horizontal axis and the fluorescence units indicated on the verticalaxis. The white bars represent the net signal from the wild-type probe,while the dark bars represent the net signal from the mutant probe. Theleft half of the graph indicates samples tested for the C282Ypolymorphism, while the right half of the graph indicates samples testedfor the H63D polymorphism. No target controls are indicated as “NT” andthe synthetic targets are indicated as SynWT and SynMT for wild-type andmutant controls, respectively. At both loci, the samples that arehomozygous for either WT or MT are easily identified by having a strongsignal from only one probe or the other, while the heterozygous samples,are easily identified by having strong signals from both the WT and MTprobes.

Example 56 Detection of Mutations in the Human MTHFR

Human 5,10-methylene-tetrahydrofolate reductase (MTHFR) is a majorenzyme in the folate-dependent regulation of methionine and homocysteineconcentrations. The wild-type protein plays a critical role in theconversion of homocysteine to methionine. A particular variation in theMTHFR protein, termed C677T and caused by a C to T transition in theMTHFR gene, has been correlated with myriad diseases and defects,including cardiovascular and neurological disorders. This Exampledescribes an assay for the detection of the WT and MT alleles for theMTHFR 677 SNP.

INVADER and probe oligonucleotide sets were designed as described aboveto detect both the WT and MT allele for the MTHFR 677 site (INVADER, WTprobe, MT probe, and FRET cassette are SEQ ID NOS:216, 217, 218 and 225,respectively). Positive control targets were synthesized for the WT andMT alleles at the 677 site (SEQ ID NOS:214 and 215, respectively);oligonucleotides are shown in FIGS. 117A and 117B.

Human genomic DNA samples 01532 and 01560 were purchased from Coriell.These samples had been characterized by “PCR” at Coriell for the MTHFRgenotype. They were also characterized in house by PCR/RFLP analysis forgenotype confirmation. Human genomic sample 32435 was purchased as wholeblood from Lampire Biological Labs., Inc. (Coopersberg, Pa.), andgenomic DNA was prepared via the Gentra PUREGENE Blood Kit (Minneapolis,Minn.) according to the manufacturer's instructions. Samples werequantitated via PicoGreen (Molecular Probes, Eugene Oreg.) and dilutedwith TE to a concentration of approximately 10 ng/μl. 100 ng (10 μl) ofeach sample was used in each reaction.

Triplicate reactions were performed and analyzed as described in Example54, except the INVADER mixes contained 1 μl of 0.5 μM INVADERoligonucleotide for each reaction. The reactions comprising thesynthetic targets as positive controls included 50 zmoles of thesynthetic target and 1 μg of yeast tRNA. Reactions simulating aheterozygous sample included 50 zmoles of each control target.

Results are shown graphically in FIG. 118, with target DNAs indicated onthe horizontal axis and the fluorescence units indicated on the verticalaxis. The white bars represent the net averaged signal from thewild-type probe, while the dark bars represent the net averaged signalfrom the mutant probe. The synthetic targets are indicated as SynWT andSynMT for wild-type and mutant controls, respectively and SynHET for amixture of the two to simulate a heterozygous sample. At both loci, thesamples that are homozygous for either WT or MT are easily identified byhaving a strong signal from only one probe or the other, while theheterozygous samples are easily identified by having strong signals fromboth the WT and MT probes.

Example 57 Detection of Mutations in the Human Prothrombin (Factor II)Gene

The prothrombin A20210G mutation has been determined to be a risk factorfor thromboembolism. The mutation occurs in the 3′ untranslated regionof the prothrombin gene, replacing the WT adenine (A) at position 20210with the MT guanine (G). This Example describes an assay for thedetection of the WT and MT alleles of the prothrombin A20210G mutation

INVADER and probe oligonucleotide sets were designed as described aboveto detect both the WT and MT alleles at the prothrombin 20210 site(INVADER, WT probe, MT probe and FRET cassette are SEQ ID NOS: 222 -225,respectively). Positive control targets were synthesized for the WT andMT alleles at the 20210 site (SEQ ID NOS:220 and 221, respectively);oligonucleotides are shown in FIGS. 119A and 119B.

Three patient samples of human genomic DNA were donated by the BloodCenter of Southeast Wisconsin, identified as 2196, 2263 and 2265. Thesesamples were purified at the Blood Center via QIAGEN BioRobot 9600(QIAGEN #900200), quantitated by Pico Green (Molecular Probes) and werefound to be at a concentration of 30-50 ng/ul.

Duplicate reactions were performed and analyzed as described in Example54, except the INVADER mixes contained 1 μl of 0.5 μM INVADERoligonucleotide for each reaction, and the mix was diluted with I volumeof dH₂O (i.e., 5 μl per reaction) so that the INVADER mix was dispensed10 μl aliquots, while the DNA was dispensed in 5 μl aliquots, adding150-250 ng of genomic DNA per reaction. Each no target control reactionreceived 1 μg of yeast tRNA, and the reactions comprising the synthetictargets as positive controls included 300 zmoles of WT control or 50zmoles of MT control and 1 μg of yeast tRNA.

Results are shown graphically in FIG. 120, with target DNAs indicated onthe horizontal axis and the fluorescence units indicated on the verticalaxis. The white bars represent the net averaged signal from thewild-type probe, while the dark bars represent the net averaged signalfrom the mutant probe. Synthetic targets are indicated as SynWT andSynMT for wild-type and mutant controls, respectively. Samples that arehomozygous for the WT are easily identified by having a strong signalfrom only the WT probe, while the heterozygous sample is easilyidentified by having strong signals from both the WT and MT probes.

Example 58 Detection of the HR-2 Mutation in the Human Factor V Gene

This Example describes an assay for the detection of the R-2polymorphism in the human factor V gene. The R-2 polymorphism in thehuman factor V gene is located in exon 13 of the factor V gene, and isthe result of an A to G transition at base 4070, replacing the wild-typeamino acid histidine with the mutant arginine in the mature protein. TheR-2 polymorphism is one of a set of mutations termed collectively HR-2.The, HR-2 haplotype is defined by 6 nucleotide base substitutions inexons 13 and 16 of the factor V gene, and is associated with anincreased functional resistance to activated protein C both in normalsubjects and in thrombophilic patients. When present as a compoundheterozygote in conjunction with the Leiden mutation (see Example 60,below), clinical symptoms are comparable to those seen in patientshomozygous for the Leiden mutation.

Within about a 600-base pair region surrounding the R2 allele, foursub-regions of DNA, each encompassing the sequence of the WT INVADERprobe set, (approximately 22 bases in length), contain sequence similarto that immediately surrounding the R2 allele. These repeated sequencescan be detected by an INVADER and probe oligonucleotide set designed forthe wild-type R-2 sequence. Because of the repeated sequence, reactionswith this INVADER/probe set yield very high signal, even with genomicsamples containing R-2 mutants, thus greatly increasing the risk ofmisinterpreting the data. In the example of an R-2 heterozygote, thesignal generated by the R-2 mutant would be extremely low compared tothe wild-type signal. It is thus possible that one might err andinterpret the data as wild-type, not as heterozygous. The same wouldhold true even for a homozygous mutant R-2 sample. Therefore, instead ofhaving an INVADER/probe set to detect the WT R-2 allele, anINVADER/probe set was developed to detect sequences in the single copya-actin gene, thus providing an internal reaction control, as well as aninternal signal intensity control. Since the α-actin gene is singlecopy, the signal levels generated in the detection of this sequence willbe comparable to that generated in the detection of the R-2 mutantallele, and the probability of incorrect data interpretation due to WTsignal overwhelming that generated by the MT is no longer an issue.

In the previous examples, each sample was assayed in two differentreactions, one reaction tested for the presence of wild-type sequenceand one reaction tested for the presence of mutant sequence. In thisexample, each sample is tested with both the α-actin internal controland the MT R-2 INVADER/probe sets. INVADER and probe oligonucleotidesets were designed as described above to detect both the MT R-2 andα-actin control sequence (MT R-2 INVADER and probe, and the FRETcassette are SEQ ID NOS:228, 229 and 230, respectively; α-actin INVADERand probe are SEQ ID NOS: 231 and 232, respectively). Positive controltargets were synthesized for the Mutant R-2 allele and the α-actin gene(SEQ ID NOS:226 and 227, respectively); oligonucleotides are shown inFIGS. 121A and B.

Human genomic DNA sample 39021 was obtained from Sigma (Catalog #D6537)and uncharacterized human genomic sample 15506 was obtained fromCoriell(Catalog #NA15506, Camden, N.J. 08103) Samples were diluted to 10ng/μl with Tris-EDTA, pH 8.0. Ten μl (100 ng) was used per reaction. Notarget controls received 1 μg of yeast tRNA instead of human genomicDNA. Reactions were performed and analyzed as described in Example 54,except the INVADER mixes contained 0.5 μl each of 2 μM R-2 INVADERoligonucleotide and 2.0 μM α-actin INVADER oligonucleotide. The probemaster mixes contained 1 μl of either the 10 μM R-2 probe or 1 μl of 10μM α-actin probe, 2 μl of 75 mM MgCl₂, 1 μl of 10 μM FRET cassette and 1μl 200 ng/μl Afu FEN-1 enzyme per reaction. The reactions comprising thesynthetic targets as positive controls included 100 zmoles of thesynthetic target and 1 μg of yeast tRNA. The SynHET and SynMT reactionscontained mixtures of synthetic targets at 2:1 and 1:1 of theα-actin:R-2 mutant targets, respectively.

Reactions were read directly on a CYTOFLUOR Multi-well Plate ReaderSeries 4000 (PerSeptive Biosystems) using the following parameters:Excitation wavelength/bandwidth 485 nm/20 nm, Emissionwavelength/bandwidth 530 nm/25nm, gain 36.

Results are shown graphically in FIG. 122, with target DNAs indicated onthe horizontal axis and the fluorescence units indicated on the verticalaxis. The white bars represent the net signal from the internal controlprobe, while the dark bars represent the net signal from the R-2 mutantprobe. Synthetic targets are indicated as SynIC and SynMT for internalcontrol and mutant R-2 controls, respectively and SynHET for a mixtureof the two to simulate a sample that is heterozygous at the R-2 allele.The sample that does not have the MT R-2 allele is easily identified byhaving a strong signal from only the IC probe, while that which isheterozygous at the R-2 allele is identified by having signals from boththe IC and the MT R-2 probes, but at a ratio near 2:1. A samplehomozygous for the mutation at the R-2 allele (not shown) would shownearly equal signal from each probe, as shown with the SynMT control.

Example 59 Detection of Single Nucleotide Polymorphisms in the HumanTNF-α Gene

The human cytokine tumor necrosis factor α (TNF-α) gene has been shownto be a major factor in graft rejection; the more TNF-α present in thesystem, the greater the rejection response to transplanted tissue. Themutation detected in this example is located in the promoter region ofthe TNF-α gene at position −308 (minus 308). The WT guanine (G) isreplaced with a MT adenine (A). This result of this promoter mutation isthe enhancement of transcription of TNF-α by 6 to 7 fold. This Exampledescribes an assay for the detection of the −308 mutation in thepromoter of the human TNF-α gene.

INVADER and probe oligonucleotide sets were designed as described aboveto detect both the WT and MT alleles at the −308 site (INVADER, WTprobe, and MT probe are SEQ ID NOS:235, 236 and 237, respectively; FRETcassette is SEQ ID NO:225). Positive control targets were synthesizedfor the WT and MT alleles at the −308 site (SEQ ID NOS:233 and 234,respectively); oligonucleotides are shown in FIGS. 123A and 123B.

Purified human genomic DNA samples (M2, M3 and M4) were donated by theMayo Clinic (Rochester Minn.).

Triplicate reactions were performed as described in Example 54, exceptthey were stopped after the four hour incubation at 63° C. by theaddition 100 μl of 100 mM EDTA. Reactions comprising the synthetictargets as positive controls included 100 zmoles of the synthetic targetand 1 μg of yeast tRNA. No target controls received 1 μg of yeast tRNAinstead of human genomic DNA. 100 μt of each stopped reaction wastransferred to a Nunc 96 well Maxisorb plate (VWR Scientific) and readon a CYTOFLUOR Multi-well Plate Reader Series 4000 (PerSeptiveBiosystems) using the following parameters: Excitationwavelength/bandwidth 485 nm/20 nm, Emission wavelength/bandwidth 530nm/25 nm; gain 65.

Results are shown graphically in FIG. 124, with target DNAs indicated onthe horizontal axis and the fluorescence units indicated on the verticalaxis. The white bars represent the net averaged signal from thewild-type probe, while the dark bars represent the net averaged signalfrom the mutant probe. The synthetic targets are indicated as SynWT andSynMT for wild-type and mutant controls, respectively and SynHET for amixture of the two to simulate a heterozygous sample. The samples thatare homozygous for either WT or MT are easily identified by having astrong signal from only one probe or the other, while the heterozygoussample is easily identified by having signals from both the WT and MTprobes.

Example 60 Detection of the Factor V Leiden Mutation

The “Leiden” mutation in blood coagulation factor V results from acytosine “C” to a thymidine “T” base change in exon 1 of the factor Vgene. The mutant protein has glutamine at amino acid position 506,instead of the wild-type arginine. This substitution prevents activatedprotein C from cleaving and inactivating factor V. The active form ofthe protein therefore remains abundant in the blood stream and continuesto promote coagulation. This Example describes an assay for thedetection of the Leiden mutation of the human factor V gene.

INVADER and probe oligonucleotide sets were designed as described aboveto detect both the WT and MT alleles at the 506 site (INVADER, WT probe,and MT probe are SEQ ID NOS:240, 241 and 242, respectively; the FRETcassette is SEQ ID NO:225). Positive control targets were synthesizedfor the WT and MT alleles at the 506 site (SEQ ID NOS:238 and 239,respectively); oligonucleotides are shown in FIGS. 125A and 125B.

Whole blood samples were obtained from the Midwest Hemostasis Center(Muncie, Ind.). Samples were characterized for the Factor V Leidengenotype by the Midwest Hemostasis Center via methods involving PCR.Buffy coats were isolated as previously described, and genomic DNA waspurified using the QIAamp 96 DNA Blood Kit (Qiagen, Valencia) accordingto the manufacturer's instructions, except that the samples were elutedin 200 μl of elution buffer. The purified DNA samples were quantitatedby Pico Green (Molecular Probes) and were then diluted with TE to aconcentration of 15-60 ng per μl. Single reactions were performed asdescribed in Example 54. The no-target control reaction received 1 μg ofyeast tRNA instead of DNA, and the reactions comprising the synthetictargets as positive controls included 200 zmoles of the synthetic targetand 1 μg of yeast tRNA.

After the 4 hour incubation at 63° C., reactions were read directly on aCYTOFLUOR Multi-well Plate Reader Series 4000 (PerSeptive Biosystems)using the following parameters: Excitation wavelength/bandwidth 485nm/20 nm, Emission wavelength/bandwidth 530 nm/25 nm, gain 65.

Results are shown graphically in FIG. 126, with target DNAs indicated onthe horizontal axis and the fluorescence units indicated on the verticalaxis. The white bars represent the net averaged signal from thewild-type probe, while the dark bars represent the net averaged signalfrom the mutant probe. The synthetic targets are indicated as SynWT andSynMT for wild-type and mutant controls, respectively and SynHET for amixture of the two to simulate a heterozygous sample. The samples thatare homozygous for either WT or MT are easily identified by having astrong signal from only one probe or the other, while the heterozygoussamples are easily identified by having signals from both the WT and MTprobes.

Example 61 Detection of Methicillin-Resistant Staphalococcus aureus

Staphylococcus aureus is recognized as one of the major causes ofinfections in humans occurring both in the hospital and in the communityat large. One of the most serious concerns in treating any bacterialinfection is the increasing resistance to antibiotics. The growingincidence of methicillin-resistant S. aureus (MRSA) infections worldwidehas underscored the importance of both early detection of the infectiveagent, and defining a resistance profile such that proper treatment canbe given. The primary mechanism for resistance to methicillin involvesthe production of a protein called PBP2a, encoded by the mecA gene. ThemecA gene is not native to Staphalococcus aureus, but is ofextra-species origin. The mecA gene is, however, indicative ofmethicillin resistance and is used as a marker for the detection ofresistant bacteria. To identify methicillin resistant S. aureus vianucleic acid techniques, both the mecA gene and at least one speciesspecific gene must be targeted. A particular species-specific gene, thenuclease or nuc gene is used in the following example. This Exampledescribes an assay for the detection of MRSA.

INVADER and probe oligonucleotide sets were designed as described aboveto detect both the mecA gene and the nuc gene (meA INVADER and probe,are SEQ ID NOS:243 and 244, respectively; nuc INVADER and probe are SEQID NOS:246 and 247, respectively; the FRET cassette for bothINVADER/probe sets is SEQ ID NO:245); oligonucleotides are shown inFIGS. 127A and 127B. The mecA and nuc target sequences shown are SEQ IDNOS:252 and 253, respectively. Samples of methicillin-resistantStaphalococcus aureus were purchased from American Type CultureCollection (ATCC, Catalog #33591). Samples of methicillin sensitiveStaphalococcus aureus (MSSA) were obtained from Gene Trak, Inc.(GT#2431), and samples of Staphalococcus haemolyticus were obtained fromATCC (ATCC#29970). The bacterial samples were streaked onto standardblood agar plates and grown at 37° C. for 14-18 hours. DNA samples fromMRSA, MSSA, and S. haemolyticus were prepared as follows. A singlecolony was suspended in 50 μl of 10 mM TRIS pH 7.5 in a 1.5 ml microfugetube. The sample was incubated at 65° C. for 5 minutes, and thenmicro-waved on the highest setting for 4 minutes. Ten μl of thispreparation was used in each reaction.

Positive controls were created by polymerase chain reaction. A 533base-pair DNA fragment of the mecA sequence and a 467 base pair DNAfragment of the nuc gene sequence were amplified and isolated asfollows. PCR primer sequences used for mecA gene amplification were5′-AAA ATC GAT GGT AAA GGT TGG C-3″ (SEQ ID NO:248)and 5′-AGT TCT GCAGTA CCG GAT TTG C-3′ (SEQ ID NO:249). PCR primer sequences used for nucgene sequence amplification were 5′-TCGCTACTAGTTGCTTAGTG-3′ (SEQ IDNO:250) and 5′-GTAAACATAAGCAACTTTAG-3′ (SEQ ID NO:251). MRSA and MSSAtarget DNA was isolated as described above. PCR reactions were doneusing the AMPLITAQ DNA Polymerase Kit with GENEAMP (PE CorporationCatalog #N808-0152). Separate reactions were done for the mecA and nucsequences, and were performed in a 100 μl final volume containing thefollowing components: 10 μl of 10×PCR buffer, 2.5 μl of 10μM upstreamprimer and downstream primer, 2 μl of 10 mM dNTP mix, 1.0 μl AMPLITAQDNA polymerase, 2 μl (10-50 ng) of bacterial DNA (the MRSA or the MSSA),and 80 μl of water for a final volume of 100 μl. Reactions were coveredwith approximately 50 μl of CHILLOUT liquid wax (MJ Research) and cycledas follows: the mecA reactions were denatured at 97° C. for 3 minutes.Reactions were then cycled at 97° C. for 1 minute, 52° C. for 30seconds, 72° C. for 1 minute. This was repeated 5 times. After the final72° C. 1 minute incubation, reactions were again heated to 94° C. for 30seconds, 52° C. for 30 seconds and 72° C. for 1 minute, for 30 cycles.mecA reactions were then incubated at 72° C. for 7 minutes, then held at4° C. until purification.

The nuc amplification reactions were denatured at 97° C. for 3 minutes.Reactions were then cycled at 97° C. for 1 minute, 48° C. for 30seconds, 72° C. for 1 minute. This was repeated for 5 cycles. After thefinal 72° C., 1 minute incubation, reactions were heated to 94° C. for30 seconds, 48° C. for 30 seconds and 72° C. for 1 minute. This wasrepeated for 30 cycles. Reactions were then incubated at 72° C. for 7minutes, and finally cooled to 4° C. and held until purification.

After amplification, reactions were run on a 1% agarose gel in 1% TBEbuffer with 50-2000 base-pair markers (Novagen,Cat#69278-3); bands werevisualized via ethidium bromide staining followed by ultravioletillumination. The appropriately sized bands were excised from the geland purified by the QIAquick Gel Extraction Kit (QiagenCat#28704)according to the manufacturer's protocol. Each column was eluted twicewith 50 μl of elution buffer. The concentration of the purified PCRsynthetic target DNA was determined by OD₂₆₀, and diluted to a stockconcentration of 50 fmoles per microliter. Positive controls were usedat a concentration of 10 amole per reaction with a 10 μl addition.Control reactions with no target were also performed, using humangenomic DNA at 10 ng/μl in place of a bacterial sample. All samples wereadded in a volume of 10 μl.

INVADER reactions were performed in MJ 96 well Low Profile PolypropyleneMicroplates (MJ Research MLL-9601) in duplicate in a final volume of 15μl. An INVADER reaction master mix was prepared and contained (perreaction) 1.5 μl water, 2.5 μl 100 mM MOPS, 5 μl of 20% PEG (8000 MW),0.5 μl of 1 μM nuc INVADER oligonucleotide and 0.5 μl of mecA INVADERoligonucleotide. Five μl of the INVADER master mix were added to eachsample and control well. 10 μl of the target bacterial DNA samples,prepared as described above, or 10 μl (10 amoles) of positive controltarget, or 10 μl (100 ng of human genomic DNA) of the no target controlsample were added to each well. Samples were overlaid with 15 μl clearChill-out wax (MJ Research,) and incubated at 95° C. for 5 minutes in anMJ Research Thermocycler with Hot Bonnet. Two different probe mastermixes were prepared, one containing the mecA probe oligonucleotide, onecontaining the nuc probe oligonucleotide. The probe master mixescontained (per reaction) 2 μl of 93.75 mM MgCl₂, 1 μl of 10μM FREToligonucleotide, 1 μl of either 10μM mecA probe oligonucleotide or 10 μMnuc probe oligonucleotide, 1 μl of 100 ng/μl Afu FEN-1 enzyme (ThirdWave Technologies, Cat #96004D). The temperature was cooled to 64° C.and 5 μl of the appropriate probe master mix (such that each control orsample reaction is tested with both the mecA and the nuc probe) wasadded below the Chill-out wax layer to each well and mixed by pipettingup and down 5 times. The plate was covered with MICROSEAL ‘A’ Film (MJResearch,) and incubated at 64° C. for 30 minutes. After the 30 minuteincubation, reactions were cooled to room temperature, placed on aPerkin Elmer MICROAMP base (cat#N801-0531) and read on a CYTOFLUORMulti-well Plate Reader Series 4000 (PerSeptive Biosystems) using thefollowing parameters: Excitation wavelength/bandwidth 485 nm/20 nm,Emission wavelength/bandwidth 530 nm/25 nm, gain 40, 30 reads per well,temperature 25° C.

Results are shown graphically in FIG. 128, with target DNAs indicated onthe horizontal axis and the fluorescence units indicated on the verticalaxis. The white bars represent the net averaged signal from the mecAprobe, while the dark bars represent the net averaged signal from thenuc probe.

Example 62 Construction of Chimerical Structure Specific Nucleases

FIG. 59 provides an alignment of the amino acid sequences of severalstructure-specific nucleases including several each of the FEN-1, XPGand RAD type nucleases. The numbers to the left of each line of sequencerefers to the amino acid residue number; portions of the amino acidsequence of some of these proteins were not shown in order to maximizethe alignment between proteins. Dashes represent gaps introduced tomaximize alignment. From this alignment, it can be seen that theproteins can be roughly divided into blocks of conservation, which mayalso represent functional regions of the proteins. While not intended asa limitation on the chimeric nucleases of the present invention, theseblocks of conservation may be used to select junction sites for thecreation of such chimeric proteins.

The Methanococcus jannaschii FEN-1 protein (MJAFEN1.PRO), the Pyrococcusfuriosus FEN-1 protein (PFUFEN1.PRO) are shown in the alignment in FIG.59. These two natural genes were used to demonstrate the creation ofchimeric nucleases having different activities than either of the parentnucleases. As known to those of skill in the art, appropriately sitedrestriction cleavage and ligation would also be a suitable means ofcreating the nucleases of the present invention. The activities of theparent nucleases on two types of cleavage structures, namely foldedstructures (See e.g., FIG. 60), and invasive structures (See e.g., FIG.26) are demonstrated in the data shown in FIGS. 129A and 129B,respectively. These test molecules were digested as described in Ex.29g. Lanes marked with “1” show cleavage by Pfu FEN-1, while lanesmarked with “2” indicate cleavage by Maj FEN-1.

In this example, PCR was used to construct complete coding sequences forthe chimeric proteins. This is a small subset of the possiblecombinations. It would also be within common practice in the art todesign primers to allow the combination of any fragment of a gene for anuclease with one or more other nuclease gene fragments, to createfurther examples of the chimeric nucleases of the present invention. Thepresent invention provides methods, including an activity test, so thatthe activity of any such chimeric nuclease not explicitly describedherein may be determined and characterized. Thus, it is intended thatthe present invention encompass any chimeric nuclease meeting therequirements of chimeric nucleases, as determined by methods such as thetest methods described herein.

To make chimeric nucleases from the M. jannaschii and P. furiosus 5′nuclease genes, homologous parts were PCR amplified using sets ofexternal and internal primers as shown in FIG. 130. In the next step, 5′portions from one gene and a 3′ portions from the other gene were joinedin pairs by recombinant PCR, such that each combination created adifferent full size chimerical gene. The resulting coding regions werecloned into the pTrc99A vector and expressed to produce chimericalnucleases. The specific details of construction of each of the chimericgenes shown in FIG. 130 are described below.

a) Construction of Chimerical 5′ Nuclease with M. jannaschii N-terminalPortion and P. furiosus C-terminal Portion With a Junction Point atCodon 84 (FIG. 130 g).

A fragment of the pTrc99A vector carrying the M. jannaschii 5′ nucleasegene was PCR amplified with TrcFwd (SEQ ID NO:266) and 025-141-02 (SEQID NO:267) primers (5 pmole each) in a 50 μl reaction using theADVANTAGE cDNA PCR kit (Clonetech), for 30 cycles (92° C., 30 s; 55° C.,1 min; 72° C. 1 min) to make an N-terminus-encoding gene fragment (SEQID NO:268). The TrcRev (SEQ ID NO:269) and 025-141-01 (SEQ ID NO:270)primers were used to amplify a fragment of the pTrc99A vector carryingthe P. furiosus gene to produce a C-terminus encoding gene fragment (SEQID NO:271). The PCR products were cleaned with the High Pure PCR ProductPurification kit (Boehringer Mannheim, Germany) as described in themanufacturer's protocol and eluted in 100 μl water.

The 025-141-02 (SEQ ID NO:267) primer and the 025-141-01 (SEQ ID NO:270)primer are complementary to each other, so that the PCR fragmentscreated above had the corresponding regions of complementarity on oneend. When these fragments are combined in an amplification reaction, theregion of complementarity allows the parts to hybridize to each other,to be filled in with the DNA polymerase, and then to be amplified usingthe outer primer pair, TrcFwd (SEQ ID NO:266) and TrcRev (SEQ ID NO:269)in this case, to form one fragment (SEQ ID NO:272). Five pmole of eachouter primer was then placed in 50 μl PCR reaction using the ADVANTAGEcDNA PCR kit (Clonetech) as described above. The full length PCR product(SEQ ID NO:272) including the chimerical coding region (positions45-1067 of SEQ ID NO:272) was separated in 1% agarose gel by standardprocedures and isolated using the Geneclean II Kit (Bio 101, Vista,Calif.). The isolated fragment was then cut with NcoI and PstIrestriction enzymes and cloned in pTrc99A vector.

b) Construction of Chimerical 5′ Nuclease with P. furiosus N-terminalPortion and M.jannaschii C-terminal Portion With a Junction Point atCodon 84 (FIG. 130 f).

A fragment of the pTrc99A vector carrying the P. furiosus 5′ nucleasegene was PCR amplified with TrcFwd (SEQ ID NO:266) and 025-141-02 (SEQID NO:267) primers (5 pmole each) as described above to make anN-terminus-encoding gene fragment (SEQ ID NO:273). The TrcRev (SEQ IDNO:269) and 025-141-01 (SEQ ID NO:270) primers were used to amplify afragment of the pTrc99A vector carrying the M. jannaschii gene toproduce a C-terminus encoding gene fragment (SEQ ID NO:274). Thefragments were purified and combined in a PCR, as described above toform one fragment (SEQ ID NO:275), containing the entire chimerical gene(positions 45-1025 of SEQ ID NO:275). This chimerical gene was cut withNcoI and PstI, and cloned into pTrc99A vector as described in a) above.

c) Construction of Chimerical 5′ Nuclease with P. furiosus N-terminalPortiono and M.jannaschii C-terminal Portion With a Junction Point atCodon 114 (FIG. 130 e).

A fragment of the pTrcPfuHis plasmid was PCR amplified with TrcFwd (SEQID NO:266) and 025-164-04 (SEQ ID NO:277) primers (5 pmole each), asdescribed above to make an N-terminus-encoding gene fragment (SEQ IDNO:276). The pTrcPfuHis plasmid was constructed by modifyingpTrc99-PFFFEN1 (described in Ex. 28), by adding a histidine tail tofacilitate purification. To add this histidine tail, standard primerdirected mutagenesis methods were used to insert the coding sequence forsix histidine residues between the last amino acid codon of thepTrc99-PFFFEN1 coding region and the stop codon. The resulting plasmidwas termed pTrcPfuHis.

The 159-006-01 (SEQ ID NO:279) and 025-164-07 (SEQ ID NO:280) primerswere used as described in section a) above, to amplify a fragment of thepTrcMjaHis plasmid to produce a C-terminus encoding gene fragment (SEQID NO:278). The pTrcMjaHis plasmid was constructed by modifyingpTrc99-MJFEN1 (described in Ex. 28), by adding a histidine tail tofacilitate purification. To add this histidine tail, standard PCRmutagenesis methods were used to insert the coding sequence for sixhistidine residues between the last amino acid codon of thepTrc99-MJFEN1 coding region and the stop codon. The resulting plasmidwas termed pTrcMjaHis. The fragments were purified, and combined by PCRamplification with TrcFwd (SEQ ID NO:266) and 159-006-01 (SEQ ID NO:279)primers in one fragment (SEQ ID NO:281) containing the chimerical gene(positions 45-1043). This chimerical gene was cut with NcoI and PstI,and cloned into pTrc99A vector as described in a), above.

d) Construction of Chimerical 5′ Nuclease with M. jannaschii N-terminalPortion and P. furiosus C-terminal Portion With a Junction Point atCodon 148 (FIG. 130 d).

A fragment of the pTrc99A vector carrying the M.jannaschii 5′ nucleasegene was PCR amplified with TrcFwd (SEQ ID NO:266) and 025-119-05 (SEQID NO:283) primers, as described above, to make an N-terminus-encodinggene fragment (SEQ ID NO:282). The TrcRev (SEQ ID NO:269) and 025-119-04(SEQ ID NO:285) primers were used to amplify a fragment of the pTrc99Avector carrying the P. furiosus gene to produce a C-terminus encodinggene fragment (SEQ ID NO:284). The fragments were purified as describedabove and combined by PCR amplification with the TrcFwd (SEQ ID NO:266)and TrcRev (SEQ ID NO:269) primers into one fragment (SEQ ID NO:286)containing the chimerical gene (positions 45-1067). This chimerical genewas cut with NcoI and PstI, and cloned into pTrc99A vector as describedin a), above.

e) Construction of Chimerical 5′ Nuclease with P. furiosus N-terminalPortion and M.jannaschii C-terminal Portion Art With a Junction Point atCodon 148 (FIG. 130 c).

A fragment of the pTrcPfuHis plasmid was PCR amplified with TrcFwd (SEQID NO:266) and 025-119-05 (SEQ ID NO:283) primers as described above tomake an N-terminus-encoding gene fragment (SEQ ID NO:287). The TrcRev(SEQ ID NO:269) and 025-119-04 (SEQ ID NO:285) primers were used toamplify a fragment of the pTrcMjaHis plasmid to produce a C-terminusencoding gene fragment (SEQ ID NO:288). The fragments were purified asdescribed above and combined by PCR amplification with TrcFwd (SEQ IDNO:266) and TrcRev (SEQ ID NO:269) primers in one fragment (SEQ IDNO:289) containing the chimerical gene (positions 45-1025). Thischimerical gene was cut with NcoI and PstI, and cloned into pTrc99Avector as described in a), above.

f) Expression and Purification of Chimeras.

All of the chimerical enzymes described above except P. furiosus-M.jannaschii construct containing a junction point at the codon 114 (i.e.,Example 62c) were purified as described for Taq DN. The P. furiosus-M.jannaschii codon 114 chimera with His-tag was purified as described forthe 5′ nuclease domain BN of Taq Pol I.

g) Activity Characterization of Natural and ChimericalStructure-Specific Nuclease.

All of the chimerical enzymes produced as described above werecharacterized. In one assay, the enzymes were tested using a mixture oflong and short hairpin substrates in the assay system described inExample 28g.

In these tests, reactions were done using 50 ng of each enzyme for 2min., at 50° C. The results of the analysis are shown in FIG. 131A. Inthis Figure, the lanes marked “1” and “2” in FIG. 131A, indicatereactions with the Pfu and Maj parent enzymes, respectively. Theremaining uncut hairpin molecules are visible as two bands at the top ofeach lane. Each chimeric enzyme tested is represented by reference inFIG. 130. For example, the lane marked “130f” shows the cleavage ofthese test molecules by the chimerical 5′ nuclease with the P. furiosusN-terminus and the M. jannaschii C-terminus joined at codon 84. Thevarious products of cleavage are seen in the lower portion of each lane.These data show that the chimerical nucleases may display cleavageactivities (i.e., substrate specificities) like either parent (e.g.,130c and parent Pfu FEN-1 show little cleavage in this test) or distinctfrom either parent (i.e., different product profiles).

Similarly, the chimerical enzymes were examined for invasive cleavageactivity using the S-60 structure and the P15 oligonucleotide depictedin FIG. 26, as described in Ex. 11. The results are shown in FIG. 131B.The uncleaved labeled P15 oligonucleotide appears in the upper portionof each lane, while the labeled product of cleavage appears in the lowerportion.

These results indicate that chimerical enzymes are different in activityand specificity from the original (i.e., wild-type) M. jannaschii and P.furiosus 5′ nucleases.

Example 63 Comparison of Digestion of Folded Cleavage Structures WithChimeric Nucleases

CFLP analysis was applied to a PCR amplified segment derived from E coli16S rRNA genes. Although bacterial 16S rRNA genes vary throughout thephylogenetic tree, these genes contain segments that are conserved atthe species, genus or kingdom level. These features have been exploitedto generate primers containing consensus sequences which flank regionsof variability. In prokaryotes, the ribosomal RNA genes are present in 2to 10 copies, with an average of 7 copies in Escherichia strains. AnyPCR amplification produces a mixed population of these genes and is inessence a “multiplex” PCR from that strain. CFLP analysis represents acomposite pattern from the slightly varied rRNA genes within thatorganism, such that no one particular rRNA sequence is directlyresponsible for the entire “bar code.” As a representative example of anamplicon as described below from the E. coli 16s rrsE gene is provided(SEQ ID NO:290). Despite the variable nature of these genes,amplification by PCR can be performed between conserved regions of therRNA genes, so prior knowledge of the entire collection of rRNAsequences for any microbe of interest is not required (See e.g., Brow etal., J. Clin. Microbiol., 34:3129 [1996]).

In this Example, the 1638 (5′-AGAGTTTGATCCTGGCTCAG-3′)(SEQ IDNO:291)/TET-1659 (5′-CTGCTGCCTCCCGTAGGAGT-3′)(SEQ ID NO:292) primer pairwas used to amplify an approximately 350 bp fragment of rrsE fromgenomic DNA derived from E. coli O157: H7 (ATCC #43895). The PCRreactions contained 10 mM Tris-HCl (pH 8.3 at 25° C.), 50 mM KCl, 1.5 mMMgCl2, 0.001% w/v gelatin, 60 μM each of dGTP, dATP, dTTP, and dCTP, 1μM of each primer, 25 ng of genomic DNA, and 2.5 units AmpliTaq DNApolymerase, LD in a volume of 100 μl. Control reactions that containedno input bacterial genomic DNA were also run to examine the amount of16S rRNA product produced due to contaminants in the DNA polymerasepreparations. The reactions were subjected to 30 cycles of 95° C. for 30sec; 60° C. for 1 min; 72° C. for 30 sec; after the last cycle the tubeswere cooled to 4° C.

After thermal cycling, the PCR mixtures were treated with E. coliexonuclease I (Exo I, Amersham) to remove single-stranded partialamplicons and primers. One unit of Exol was added directly to each PCRmixture, and the samples were incubated at 37° C. for 20 minutes. Then,the nuclease was inactivated by heating to 70° C. for 15 min. Thereaction mixtures were brought to 2 M NH₄OAc, and the DNAs wereprecipitated by the addition of 1 volume of 100% ethanol.

Cleavage reactions comprising 1 μl of TET-labeled PCR products(approximately 100 fmoles) in a total volume of 10 μl containing 1×CFLPbuffer (10 mM MOPS, pH 7.5; 0.5% each Tween 20 and NP-40) and 0.2 mMMnCl₂, were then conducted. All components except the enzyme wereassembled in a volume of 9 μl. The reactions were heated to 95° C. for15 sec., cooled to 55° C., and the cleavage reactions were started bythe addition of 50 ng of enzyme. After 2 minutes at 55° C., thereactions were stopped by the addition of 6 μl of a solution containing95% formamide, 10 mM EDTA and 0.02% methyl violet.

Reaction mixtures were heated at 85° C. for 2 min, and were thenresolved by electrophoresis through a 10% denaturing polyacrylamide gel(19:1 cross link) with 7M urea in a buffer of 45 mM Tris-Borate, pH 8.3,1.4 mM EDTA, and were visualized using the FMBIO-100 Image Analyzer(Hitachi). The resulting scanned image is shown in FIG. 132. In thisFigure, the enzymes used in each digest are indicated at the top of eachlane. CLEAVASE BN is described in Ex. 2. Lane 2 shows the results ofdigestion with the Mja FEN-1 parent nuclease, while digests with thechimerical nucleases are indicated by reference to the diagrams in FIG.130. These data show that the use of each of these nucleases underidentical reaction conditions (i.e., conditions in which the DNA assumessimilar folded structures) can produce distinct pattern differences,indicating differences in the specificities of the enzymes. Thus, eachenzyme can provide additional information about the folded structureassumed by a nucleic acid of interest, thereby allowing more accuratecomparisons of molecules for identification, genotyping, and/or mutationdetection.

These data show that the activities of these enzymes may varysubstantially in similar reaction situations. The performance of anoptimization panel for an unknown enzyme can help in selection of theoptimal enzyme and conditions for a given application. For example, inthe invasive cleavage reactions it is often desirable to choose acombination of nuclease and conditions that perform invasive cleavage,but that do not exhibit activity in the absence of the invaderoligonucleotide (i.e., do not cut a hairpin type substrate). Theoptimization panel allows selection of conditions that do not favorhairpin cleavage, such as the use of the Pfu FEN-1 enzyme in aMgCl₂-containing solution. Conversely, hairpin cleavage is desirable forCFLP-type cleavage, so it is contemplated that reaction conditions bescreened accordingly for strength in this activity.

Example 64 Characterization of Performance of Structure-SpecificNucleases

Two substrates were used to determine the optimal conditions for sevenenzymes, Afu, Pfu, Mth and Mja FEN-1s, CLEAVASE BN, Taq DN and Tth DN.As shown in FIG. 140 Panel A, Substrate 25-65-1(5′-Fluorescein-TTTTCGCTGTCTCGCTGAAAGCGAGACAGCGTTT-3′; SEQ ID NO:293) isa stem-loop structure with a 5′ arm labeled at its 5′ end withfluorescein. As shown in FIG. 140 Panel B, substrate 25-184-5(INVADER-like “IT” test substrate”)(5′-Fluorescein-TTTTCGCTGTCTCGCTGAAAGCGAGACAGCGAAAGACGCTCGTGAAACGAGCGTCTTTG-3′; SEQ ID NO:294) is a substrate with an upstream primeradjacent to the 5′ fluoroscein labled arm; this mimics an INVADERoligonucleotide and target (“IT”). Standard reactions contained 2 μMlabeled substrate, 10 mM MOPS, pH7.5, 0.05% TWEEN 20, 0.05% NP-40, 20μg/ml tRNA (Sigma #R-5636) and 2 mM MgCl2 or 2 mM MnCl2. Ten μlreactions were heated to 90° C. for 15 seconds in the absence of enzymeand divalent cation, after which the reactions were cooled to roomtemperature and enzyme was added. Reactions were heated to 50° C. for 20seconds and divalent cation was then added to start the reaction. Theincubation time varied from 1 minute to 1 hour depending on theparticular enzyme/substrate combination. Reaction times were adjusted sothat less than 25% of the substrate was cleaved during the incubation.Reactions were stopped with the addition of 10 μl of 95% formamide, 20mM EDTA, methyl violet. One μl of each reaction was electrophoresed on a20% denaturing acrylamide gel and then scanned on an FMBIO 100 scanner(Hitachi).

Divalent cation titrations varied MgCl₂ or MnCl₂ from 0.25 mM to 7 mMunder otherwise standard conditions. Salt titrations varied KCl from 0mM to 200 mM or 400 mM for salt tolerant enzymes under otherwisestandard conditions. For temperature titrations, reactions with CLEAVASEBN and the FEN-1 enzymes contained 50 mM KCl and 4 mM MgCl₂ or MnCl₂.Temperature titrations with Taq DN and Tth DN contained 200 mM KCl and 4mM MgCl₂ or MnCl₂. Temperature was varied from 40° C. to 85° C. in 5 or10 degree increments depending on the particular enzyme used.

The results are shown in FIGS. 133-139. FIG. 133 shows the results forCLEAVASE BN, while FIG. 134 shows the results for Taq DN, FIG. 135 showsthe results for Tth DN, FIG. 136 shows the results for Pfu FEN-, FIG.137 shows the results for Mja FEN-, FIG. 138 shows the results for AftiFEN-1, and FIG. 139 shows the results for Mth FEN-1. In each of thePanels within these Figures, the activity of the enzyme is defined ascleavages per molecule of enzyme per minute. Panels marked “IT” refer tocleavage of the 25-184-5 structure (SEQ ID NO:294; FIG. 140B), whichmimics an INVADER oligo/target DNA structure, while Panels marked with“hairpin” refer to cleavage of the 25-65-1 structure (SEQ ID NO:293;FIG. 140A), which indicates activity on folded cleavage structures.

In each of these Figures, Panel A shows the results from reactionscontaining 2 mM MgCl₂ and the IT substrate as described in the text,with KCl varied as indicated; Panel B shows the results from reactionscontaining 2 mM MnCl₂ and the IT substrate as described in the text,with KCl varied as indicated; Panel C shows the results from reactionscontaining 2 mM MgCl₂ and the hairpin substrate as described in thetext, with KCl varied as indicated; Panel D shows the results fromreactions containing 2 mM MnCl₂ and the hairpin substrate as describedin the text, with KCl varied as indicated; Panel E shows the resultsfrom reactions containing the IT substrate as described in the text,with MgCl₂ varied as indicated; Panel F shows the results from reactionscontaining the IT substrate as described in the text, with MnCl₂ variedas indicated; Panel G shows the results from reactions containing thehairpin substrate as described in the text, with MgCl₂ varied asindicated; Panel H shows the results from reactions containing thehairpin substrate as described in the text, with MnCl₂ varied asindicated; Panel I shows the results from reactions containing the ITsubstrate, 4 mM MgCl₂, and 50 mM KCl (Afu FEN-1, Pfu FEN-1, Mja FEN-1,Mth FEN-1, and CLEAVASE FN) or 200 mM KCl (Taq DN and Tth DN) asdescribed in the text, with the temperature varied as indicated; andPanel J shows the results from reactions containing the IT substrate, 4mM MnCl2, and 50 mM KCl (Afli FEN-1, Pfu FEN-1, Mja FEN-1, Mth FEN-1,and CLEAVASE BN) or 200 mM KCl (Taq DN and Tth DN) as described in thetext, with the temperature varied as indicated. It is noted that some ofthese FIGS (e.g., 134, 135, 136, and 138) do not show each of theabove-named panels A-J.

From the above it is clear that the invention provides reagents andmethods to permit the detection and characterization of nucleic acidsequences and variations in nucleic acid sequences. The INVADER-directedcleavage reaction of the present invention provides an ideal directdetection method that combines the advantages of the direct detectionassays (e.g., easy quantification and minimal risk of carry-overcontamination) with the specificity provided by a dual or trioligonucleotide hybridization assay.

Example 65 Cloning and Expression of Unknown FEN1 Nucleases

A common method for cloning new members of a gene family is to run PCRreactions using degenerate oligonucleotides complementary to conservedamino acid sequences in that family, and then to clone and sequence thegene-specific PCR fragments. This sequence information can then be usedto design sense and anti-sense gene-specific primers which can be usedin PCR walking reactions (Nucleic Acids Res. 1995a. 23(6)1087-1088) toobtain the remainder of the gene sequence. The sequences obtained fromthe sense and anti-sense PCR walks can then be combined to generate theDNA sequence for the entire open reading frame (ORF) of the gene ofinterest. Once the entire ORF is know, primers specific to both the 5′and the 3′ end of the gene can be designed, and PCR reactions can beperformed on genomic DNA to amplify the gene in its entirety. Thisorganism-specific, amplified fragment can then be cloned into anexpression vector, and via methods know in the art, and detailed below,the protein of interest can be expressed and purified.

The following examples utilize this series of steps in the cloning andexpression of 14 novel FEN-1 nucleases. The steps and reagents (such ascloning vectors, expression vectors, PCR kits or PCR walking kits, etc)are intended to be examples and not limitations to the currentinvention; those skilled in the art would know that different cloningvectors, expression vectors, PCR kits or PCR walking kits, etc. may besubstituted for those exemplified.

The following example is divided into 2 main sections:

-   A. degenerate PCR and PCR walking to obtain the sequence of 14 novel    FEN-1 nucleases-   B. cloning and expression of 16 FEN-1 nucleases    A. Degenerate PCR and PCR Walking to Obtain the Sequence of 14 Novel    FEN-1 Nucleases

The protein sequences of the FEN1 genes from Pyrococcus furiosus (SEQ IDNO:79) Methanococcus jannaschii (SEQ ID NO:75), Methanobacteriumthermoautotrophicum (SEQ ID NO:265), and Archaeoglobus fulgidus (SEQ IDNO:165) were aligned and blocks of conserved amino acids wereidentified. The conserved sequence blocks VFDG (valine, phenylalanine,aspartic acid, glycine), EGEAQ (glutamic acid, glycine, glutamic acid,alanine, glutamine), SQDYD (serine, glutamine, aspartic acid, tyrosine,aspartic acid), and GTDYN/GTDFN (glycine, threonine, aspartic acid,tyrosine or phenylalanine, asparagine) were chosen as sequences thatwould likely be present in all Archaeal FEN1 genes. Degenerateoligonucleotides were designed for each of these conserved sequenceblocks. In addition to the FEN1 gene specific portion of theoligonucleotides a 15 nucleotide tail was added to the 5′ end of theoligonucleotides to enable nested PCR. A different tail sequence wasused depending on whether the degenerate oligonucleotide targets thesense or antisense strand of the FEN1 gene.

Forward and/or reverse versions of the oligonucleotides were made andtarget the sense and antisense strands of the FEN1 gene respectively.The oligonucleotides are VFDG-Fwd (SEQ ID NO:295), EGEAQ-Fwd (SEQ IDNO:296) QDYD-Fwd (SEQ ID NO:297), EGEAQ-Rev (SEQ ID NO:298), SQDYD-Rev1(SEQ ID NO:299), SQDYD-Rev2 (SEQ ID NO:300), and GTDYN-Rev (SEQ IDNO:301). Two oligonucleotides were made for the SQDYD-Rev sequencebecause serine is encoded by 6 different codons. For use in PCR, theSQDYD-Rev1 and SQDYD-Rev2 oligonucleotides were mixed in a ratio of 1:2.For the QDYD-Fwd oligonucleotide, the requirement for mixing was avoidedby targeting only the last four amino acids of the conserved SQDYDsequence. The GTDYN-Rev oligonucleotide also recognizes the sequenceGTDFN since the codons for tyrosine and phenylalanine share 2 of 3nucleotides.

First, genomic DNA was prepared from 1 vial of the live bacterial strainas described below. All bacterial strains were obtained from the DSMZ(Deutsche Sanimlung von Mikroorganismen und Zellkulturen, Acidianusambivalens—DSM #3772). When the cells were lyophilized, they wereresuspended in 200 μl of TNE (10 mM TrisHCL, pH 8.0, 1 mM EDTA, 100 mMNaCl). When the cells were in liquid suspension, they were spun down at20,000×G for 2 minutes and the cell pellets were resuspended in 200 μlof TNE. 20 μl of 20% SDS (sodium dodecylsulfate) and 2 μl of 1 mg/mlproteinase K were added and the suspension was incubated at 65° C. for30 minutes. The lysed cell suspension was extracted in sequential orderwith buffered phenol, 1:1 phenol: chloroform, and chloroform. Thenucleic acid was precipitated by the addition of on equal volume of cold100% ethanol. The nucleic acid was pelleted by spinning at 20,000×G for5 minutes. The nucleic acid pellet was washed with 70% ethanol, airdryed and resuspended in 50 μl of TE (10 mM TrisHCL, pH 8.0, 1 mM EDTA).The final DNA pellet was re-suspended in 50 μl of TE (10 mM Tris HCl, pH8.0, 1 mM EDTA).

Both reactions of the nested PCR were done using the Advantage cDNA PCRkit (Clontech) according to manufacturer's instructions using a finalconcentration of 1 μM for all oligonucleotides. The first reaction isdone in a 20 μl volume with one of the 6 possible combinations offorward and reverse degenerate oligonucleotides, and includes either 1μl of the genomic DNA preparation described above, or in the case of Tgoand Tzi, 1 μl (4 ng/μl stock and 6 ng/μl stock, respectively) of DNApurchased from ATCC (ATCC#s 700654D and 700529D, respectively). Thecycling conditions were 20 cycles of 95° C. for 15 seconds, 50° C. or55° C. for 15 seconds, and 68° C. for 30 seconds. The second reactionsutilize primers that have the same sequence as the 5′ tail sequence ofthe degenerate oligonucleotides described above. The two primers are203-01-01 (SEQ ID NO:302) and 203-01-02 (SEQ ID NO:303). The secondreaction is carried out exactly as described for the first reaction,except 30 cycles are done instead of 20 and the reaction volume is 25μl. Following the second PCR, 5 pi of the reaction were loaded on a 2%or 4% agarose gel and the DNA was visualized by ethidium bromidestaining. The expected product sizes based on the previously identifiedFEN1 sequences for all primer pairs are as follows: VFDG-Fwd andEGEAQ-Rev; 275 base pairs, VFDG-Fwd and SQDYD-Rev; 325 base pairs, VFDGFwd and GTDYN-Rev; 510 base pairs, EGEAQ-Fwd and SQDYD-Rev; 100 basepairs, EGEAQ-Fwd and GTDYN-Rev; 290 base pairs, QDYD-Fwd and GTDYN-Rev;230 base pairs. The primer pair, VFDG-Fwd and EGEAQ-Rev was able togenerate a correctly sized DNA product for all samples attempted. Theprimer pair, VFDG-Fwd and GTDYN-Rev was able to generate a correctlysized DNA product for most of the DNA samples attempted.

When a DNA product of the expected size was made by the degenerate PCR,that DNA fragment was isolated and cloned into pGEM-T Easy (Promega)using the pGEM-T Easy ligation kit according to the manufacturer'sinstructions. The DNA sequence was determined and the sequence was usedto generate sense and antisense genome walking oligonucleotides forcloning the remainder of the FEN1 genes. The oligonucleotides weredesigned according to the parameters of the GenomeWalker kit (Clontech)which was used prepare the various genomic DNA samples for the genomewalking PCR reactions.

Since many of the organisms of interest cannot be easily cultured in astandard laboratory setting (due to requirements of very specializedtemperature, pressure and medium conditions), quantities of genomic DNAwere limiting. Therefore, the DNA was randomly amplified using a random12-mer-oligonucleotide. One hundred-μl PCR reactions were set up withthe Advantage cDNA PCR kit (Clontech) and contained 10 μl of genomic DNAand 15 μM random 12-mer oligonucleotide. 50 cycles were carried out withthe following parameters: 95° C. for 30 seconds, 50° C. for 30 seconds,68° C. for 5 minutes. After the PCR reactions were complete, amplifiedDNA was purified with the High Pure PCR Product Purification kit(Boehringer Mannheim). The purified DNA was eluted into a total of 200μl of 10 mM TrisHCL, pH 8.5.

The genome walking protocol consists of 3 steps. First, a genomic DNAsample is cut with 5 different blunt-end restriction enzymes in 5separate reactions. Second, the cut DNA is ligated to an adapter thatserves as a tag sequence and also is designed to prevent backgroundamplification. Third, the ligated DNA is amplified with a gene-specificprimer and a primer with the same sequence as a portion of the adaptersequence. 50 μl restriction digests contained 30 μl of randomlyamplified genomic DNA and one of the following enzymes: Dra I, Eco RV,Pvu II, Sca I or Stu I. After 4 hours at 37° C., the cut DNA waspurified with either GENECLEANII (Bio 101) or QIAEX II (Qiagen)according to manufacturer's instructions. DNA was eluted into 10 μl of10 mM TrisHCl, pH 8.5 in either case. 5.6 μl of this cut DNA was used in10 μl ligation reactions containing 6 μM GenomeWalker adapter. Reactionswere carried out at room temperature overnight followed by heating at70° C. for 10 minutes to inactivate the T4 DNA ligase. The ligationreactions were then diluted with 70 μl of TE (10 mM TrisHCl, pH 8.0, 1mM EDTA).

One μl of the diluted ligation mix was used in 25 μl PCR reactions with0.2 μM gene-specific primer and 0.2 μM primer AP-1 (Clontech) which hasthe same sequence as the 5′ portion of the GenomeWalker adapter. Tenreactions were done for each DNA sample. Five antisense walk PCRreactions (for the 5 different restriction enzymes used to cut thegenomic sample) were done using the sense gene-specific primer and fivesense walk PCR reactions were done using the antisense gene-specificprimer for each DNA sample. The cycling parameters were as recommendedby the Universal Genome Walking kit (Clontech) and were as follows: 7cycles of 94° C. for 25 seconds and 72° C. for 3 minutes, 32 cycles of94° C. for 25 seconds and 67° C. for 3 minutes, followed by 67° C. for 7minutes. The source of DNA for the genome walking PCR reactions was, inmost cases, genomic DNA which had been randomly amplified with a random12-mer oligonucleotide, as described above. The exceptions were forSulfolobus solfataricus (Sso), Thermococcus gorgonarius (Tgo), andThermococcus zilligii (Tzi). Because we were able to grow, Sso, therewas a large quantity of Sso genomic DNA, and the DNA was used directly.A 500 ml culture of Sulfolobus solfataricus (ATCC #35091) was grown inDSM medium 182 at 75 C for 48 hours. After growth, the cells were spundown for 10 minutes at 20,000×G at 4 C. The cell pellet was resuspendedin 10 ml of TE (10 mM Tris HCL, 1 mM EDTA) and frozen in 1 ml aliquotsat −70 C. DNA was prepared by the method described above using 1 mlaliquots, but all volumes were increased 10-fold. As noted above, theTgo and Tzi genomic DNAs were purchased from ATCC.

After the PCR reactions were completed, 5 μl of each reaction was run ona 1% agarose gel and the DNA was visualized by ethidium bromidestaining. The presence of a major product was used as an indication of asuccessful reaction. When a major product was made, it was gel purifiedwith GENECLEAN II (Bio101) or QIAEX II (Qiagen) and ligated into pGEM-TEasy (Promega) according to manufacturer's instructions. The plasmidswere sequenced with primers flanking the insert and the sequenceobtained was compared to the sequence of the fragment generated by PCRwith degenerate oligonucleotides from the same species. The sequencesobtained for the degenerate PCR and the sense and antisense walks werecombined to generate the DNA sequence for the entire FEN1 open readingframe. Specific information regarding genome walking and cloning foreach of the 14 novel FEN-1 nucleases is detailed below.

1. Acidianus ambivalens (Aam)

The Acidianus ambivalens (Aam) genome walks were done as follows. Theantisense primer was Aam 39AS (SEQ ID NO:304) and the sense primer wasAam 44S (SEQ ID NO:305). The antisense PCR walk on Sca I digested Aamgenomic sample generated a 1 kilobase DNA product which was cloned intopGEM-T Easy (Promega) following manufacturer's instructions andsequenced. The sense PCR walk on Dra I digested Aam genomic samplegenerated a 600 base pair fragment which was cloned into pGEM-T Easy(Promega) following manufacturer's instructions and sequenced.

2. The Acidianus brierlyi (Abr)

The Acidianus brierlyi (Abr) genome walks were done as follows. Theantisense primer was Abr 39AS (SEQ ID NO:306) and the sense primer wasAbr 40S (SEQ ID NO:307). The antisense PCR walk on Eco RV digested Abrgenomic sample generated a 1.5 kilobase DNA product which was clonedinto pGEM-T Easy (Promega) following manufacturer's instructions andsequenced. The sense PCR walk on Dra I digested Abr genomic samplegenerated a 600 base pair fragment which was cloned into pGEM-T Easy(Promega) following manufacturer's instructions and sequenced.

3. Archaeoglobus profundus (Apr)

The Archaeoglobus profundus (Apr) genome walks were done as follows. Theantisense primer was Apr 35AS (SEQ ID NO:308) and the sense primer wasAbr 63S (SEQ ID NO:309). The antisense PCR walk on Dra I digested Aprgenomic sample generated a 1.8 kilobase DNA product which was clonedinto pGEM-T Easy (Promega) following manufacturer's instructions andsequenced. The antisense PCR walk on Pvu II digested Apr genomic samplegenerated a 2 kilobase DNA product which was cloned into pGEM-T Easy(Promega) following manufacturer's instructions and sequenced. The sensePCR walk on Dra I digested Apr genomic sample generated a 1 kilobasefragment which was cloned into pGEM-T Easy (Promega) followingmanufacturer's instructions and sequenced.

4. Archaeoglobus veneficus (Ave)

The Archaeoglobus veneficus (Ave) genome walks were done as follows. Theprimary antisense primer was Ave 34AS (SEQ ID NO:310) and the primarysense primer was Ave 65S (SEQ ID NO:311). For Ave, the primary genomewalk PCR reactions generated a strong product for only the sense walk onthe Dra I cut Ave DNA sample. Therefore, nested PCR reactions were doneusing the nested primer AP-2 and either the nested antisense primer Ave32AS (SEQ ID NO:312) or the nested sense primer Ave 67S (SEQ ID NO:313).25 μl nested reactions were done as described above for the primary PCRwalk reactions. The primary reactions were diluted 1:50 in H₂O and 0.5μl of those dilutions were added to the nested PCR reactions. Thecycling parameters for the nested PCR reactions were as recommended bythe Universal Genome Walking kit (Clontech) and are as follows: 5 cyclesof 94° C. for 25 seconds and 72° C. for 3 minutes, 20 cycles of 94° C.for 25 seconds and 67° C. for 3 minutes, followed by 7 minutes at 67° C.The nested antisense PCR reaction on Stu I cut Ave genomic samplegenerated a 1 kilobase DNA product which was cloned into pGEM-T Easy(Promega) following manufacturer's instructions and sequenced. Thenested sense PCR reaction on Eco RV cut Ave genomic sample generated a1.1 kilobase product which was cloned into pGEM-T Easy (Promega)following manufacturer's instructions and sequenced.

5. Desulfurococcus amylolyticus (Dam)

The Desulfurococcus amylolyticus (Dam) genome walks were done asfollows. The antisense primer was Dam 31AS (SEQ ID NO:314) and the senseprimer was Dam 65S (SEQ ID NO:315). The antisense PCR walk on Stu Idigested Dam genomic sample generated a 1 kilobase DNA product which wascloned into pGEM-T Easy (Promega) following manufacturer's instructionsand sequenced. The sense PCR walk on Pvu II digested Dam genomic samplegenerated a 800 base pair DNA product which was cloned into pGEM-T Easy(Promega) following manufacturer's instructions and sequenced. The sensePCR walk on Stu I digested Dam genomic sample generated a 400 base pairfragment which was cloned into pGEM-T Easy (Promega) followingmanufacturer's instructions and sequenced.

6. Desulfurococcus mobilis (Dmo)

The Desulfurococcus mobilis (Dmo) genome walks were done as follows. Theantisense primer was Dmo 31AS (SEQ ID NO:316) and the sense primer wasDmo 66S (SEQ ID NO:317). The antisense PCR walk on Eco RV digested Dmogenomic sample generated a 450 base pair DNA product which was clonedinto pGEM-T Easy (Promega) following manufacturer's instructions andsequenced. The sense PCR walk on Pvu II digested Dmo genomic samplegenerated a 1 kilobase fragment which was cloned into pGEM-T Easy(Promega) following manufacturer's instructions and sequenced.

7. Methanococcus igneus (Mig)

The Methanococcus igneus (Mig) genome walks were done as follows. Theantisense primer was Mig 36AS (SEQ ID NO:318) and the sense primer wasMig 39S (SEQ ID NO:319). The antisense PCR walk on Dra I digested Miggenomic sample generated a 900 base pair DNA product which was clonedinto pGEM-T Easy (Promega) following manufacturer's instructions andsequenced. The sense PCR walk on Eco RV digested Mig genomic samplegenerated a 2.5 kilobase fragment which was cloned into pGEM-T Easy(Promega) following manufacturer's instructions and sequenced.

8. Methanopyrus kandleri (Mka)

The Methanopyrus kandleri (Mka) genome walks were done as follows. Theantisense primer was Mka 31AS (SEQ ID NO:320) and the sense primer wasMka 41S (SEQ ID NO:321). The antisense PCR walk on Eco RV digested Mkagenomic sample generated a 500 base pair DNA product which was clonedinto pGEM-T Easy (Promega) following manufacturer's instructions andsequenced. The sense PCR walk on Eco RV digested Mka genomic samplegenerated a 1.6 kilobase fragment which was cloned into pGEM-T Easy(Promega) following manufacturer's instructions and sequenced.

9. Pyrobaculum aerophilum (Pae)

The Pyrobaculum aerophilum (Pae) genome walks were done as follows. Theantisense primer was Pae 28AS (SEQ ID NO:322) and the sense primer wasPae 45S (SEQ ID NO:323). The antisense PCR walk on Eco RV digested Paegenomic sample generated a 400 base pair DNA product which was clonedinto pGEM-T Easy (Promega) following manufacturer's instructions andsequenced. The sense PCR walk on Pvu II digested Pae genomic samplegenerated a 700 base pair fragment which was cloned into pGEM-T Easy(Promega) following manufacturer's instructions and sequenced.

10. Pyrodictium brockii (Pbr)

The Pyrodictium brockii (Pbr) genome walks were done as follows. Theantisense primer was Pbr 42AS (SEQ ID NO:324) and the sense primer wasPbr 56S (SEQ ID NO:325). The antisense PCR walk on Eco RV digested Pbrgenomic sample generated a 650 base pair DNA product which was clonedinto pGEM-T Easy (Promega) following manufacturer's instructions andsequenced. The sense PCR walk on Pvu II digested Pbr genomic samplegenerated a 800 base pair fragment which was cloned into pGEM-T Easy(Promega) following manufacturer's instructions and sequenced.

11. Sulfolobus soalftaricus (Sso)

The Sulfolobus solfataricus (Sso) genome walks were done as follows. Theantisense primer was Sso 27AS (SEQ ID NO:326) and the sense primer wasSso 27S (SEQ ID NO:327). The antisense PCR walk on Pvu II digested Ssogenomic DNA generated a 1 kilobase DNA product which was cloned intopGEM-T Easy (Promega) following manufacturer's instructions andsequenced. The sense PCR walk on Dra I digested Sso genomic DNAgenerated a 750 base pair fragment which was cloned into pGEM-T Easy(Promega) following manufacturer's instructions and sequenced.

12. Thermococcus gorgonarius (Tgo)

The Thermococcus gorgonarius (Tgo) genome walks were done as follows.The antisense primer was Tgo 55AS (SEQ ID NO:330) and the sense primerwas Tgo 67S (SEQ ID NO:331). The antisense PCR walk on Dra I digestedTgo genomic sample generated a 1 kilobase DNA product which was clonedinto pGEM-T Easy (Promega) following manufacturer's instructions andsequenced. The sense PCR walk on Stu I digested Tgo genomic samplegenerated a 850 base pair fragment which was cloned into pGEM-T Easy(Promega) following manufacturer's instructions and sequenced.

13. Thermococcus litoralis (Tli)

The Thermococcus litoralis (Tli) genome walks were done as follows. Theantisense primer was Tli 28AS (SEQ ID NO:328) and the sense primer wasTli 48S (SEQ ID NO:329). The antisense PCR walk on Eco RV digested Tligenomic sample generated a 1 kilobase DNA product which was cloned intopGEM-T Easy (Promega) following manufacturer's instructions andsequenced. The sense PCR walk on Eco RV digested Tli genomic samplegenerated a 1.9 kilobase fragment which was cloned into pGEM-T Easy(Promega) following manufacturer's instructions and sequenced.

14. Thermococcus zilligii (Tzi)

The Thermococcus zilligii (Tzi) genome walks were done as follows. Theantisense primer was Tzi 55AS (SEQ ID NO:332) and the sense primer wasTzi 67S (SEQ ID NO:333). The antisense PCR walk on Dra I digested Tzigenomic sample generated a 1 kilobase DNA product which was cloned intopGEM-T Easy (Promega) following manufacturer's instructions andsequenced. The sense PCR walk on Stu I digested Tzi genomic samplegenerated a 850 base pair fragment which was cloned into pGEM-T Easy(Promega) following manufacturer's instructions and sequenced.

B. Cloning of 16 FEN-1 Nucleases

The previous section detailed the cloning and'sequencing of novel FEN-1nucleases. This section will describe the cloning of these novel FEN-1's into an expression vector, as well as the cloning of two additional,previously known FEN-1 's, Aeropyrnum pemix (Ape) and Pyrococcushorikoshii (Pho). The process comprises either 4 or 5 steps, and isbroadly outlined below.

The first step involves the design of 5′ and 3′ gene specific PCRprimers. Since the complete sequence of all 16 FEN-1 genes has beendetermined, specific primers were designed to target and amplify theentire FEN-1 gene of interest. For each FEN1 endonuclease to be cloned,the 5′-end primer and the 3-end primer are mostly complementary to the5′ end and the 3′ end of the FEN1 open reading frame. The first fewnucleotides of the primer constitute a spacer and sequences necessary tointroduce a restriction endonuclease site to facilitate cloning. Thechoice of restriction endonuclease sequence to incorporate into theprimer is dependent on the sequence of the FEN-1 enzyme. Ideally, uniquerestriction sites are created at the 5′ and 3′ ends of the PCR product(they are not the same as any restriction sites that may be internal tothe FEN-1 sequence). Also, the sites incorporated into the primerscorrespond to restriction enzyme sites that are commonly found onexpression vectors used in the art. Examples of such enzymes are EcoRi,Sall, NcoI, XbaI and PstI.

Second, PCR reactions were performed using the primers designed aboveand genomic DNA from the organism of interest. Third, the PCR productswere gel purified and then cut with restriction endonucleasescorresponding to the sites incorporated in the PCR primers. The cut PCRproducts were then purified away from the smaller digest fragments and,fourth, these cut products were cloned into an expression vector. Insome cases, this was the final step of the cloning process, prior totransformation and protein expression/purification. In some cases afifth step was needed. In some cases, a mutagenesis step had to beperformed to remove any nucleotides that were incorporated into the ORFas a result of primer sequences required for cloning.

Finally, a bacterial host (e.g., E. coli JM109) was transformed with theexpression vector containing the cloned FEN-1, and protein expressionand purification were done as detailed in Experimental Example 28f. Fourof the isolated FEN gene constructs (Pae FEN-1, Pbr FEN-1, Mig FEN-1 andMka FEN-1) produced little or no detectable protein in the host that wastransformed.

The following section details the PCR, restriction digests, cloning,mutagenesis reactions (if required) and transformation for each FEN-1nuclease. The description is sub-divided in order to group the FEN-1nucleases according to the restriction endonucleases used in cloning.The sub-divisions are as follows:

-   I. FEN-1 endonucleases cloned with restriction endonucleases    NcoI/SalI-   II. FEN-1 endonucleases cloned with restriction endonucleases    EcoRI/SalI-   III. FEN-1 endonucleases cloned with other restriction endonucleases    I. Cloning of FEN-1 Endonucleases Using the Restriction    Endonucleases NcoI and SalI.

In this example, DNA encoding the FEN-1 endonuclease from Acidianusambivalens (Aam), Acidianus brierlyi (Abr), Aeropyrum pernix (Ape),Archaeaglobus profundus (Apr), Methanococcus igneus (Mig), Pyrococcushorikoshii (Pho), Sulfolobus solfataricus (Sso), Thermococcusgorgonarius (Tgo), were isolated and inserted into a plasmid under thetranscriptional control of an inducible promoter as follows.

1. Cloning of Acidianus ambivalens FEN-1 (Aam)

One microliter of the genomic DNA solution described above (in section65A) was employed in a PCR using the ADVANTAGE cDNA PCR kit (Clonetech);the PCR was conducted according to manufacturer's recommendations. Foreach FEN1 endonuclease to be cloned, the 5′-end primer is mostlycomplementary to the 5′ end of the FEN1 open reading frame. The first 6nucleotides of the primer constitute a spacer and 2 bases of an Nco Isite to facilitate cloning. An ‘A’ at position 3 of the Aam ORF sequence(SEQ ID NO:336) was mutated to a ‘G’ in the 5′ primer to create an ATPstart codon. Likewise, the 3′-end primer is mostly complementary to the3′ end of the FEN-1 open reading frame. The first 10 nucleotidesconstitute a spacer and Sal I site to facilitate cloning. The PCRprimers used for Aam are: Aam5′-5′ GCAACCATG GGAGTAGACCTTGCTGATTTGG (SEQID NO:334)and Aam 3′-5′CCATGTCGACTAAAACCACTGATCTAAACCGC (SEQ ID NO:335).The PCR reaction for each FEN1 resulted in the amplification (i.e.production) of a single major band about 1 kilobase in length. The openreading frame (ORF) encoding the Aam FEN-1 endonuclease is provided inSEQ ID NO:336; the amino acid sequence encoded by the Aam ORF isprovided in SEQ ID NO:337.

Following the PCR amplification, the entire reaction was electrophoresedon a 1.0% agarose gel and the major band was excised from the gel andpurified using the GENECLEAN II kit (Bio 101, Vista Calif.) according tomanufacturer's instructions. Approximately 1 μg of the gel-purifiedFEN-1 PCR product was digested with NcoI and SalI. After digestion, theDNA was purified using the Geneclean II kit according to manufacturer'sinstructions. One microgram of the pTrc99a vector (Pharmacia) wasdigested with NcoI and SalI in preparation for ligation with thedigested PCR product. One hundred nanograms of digested pTrc99a vectorand 250 ng of digested FEN-1 PCR product were combined and ligated tocreate pTrc99-(enzyme TLA)FEN1. pTrc99-(enzyme TLA) FEN-1 was used totransform competent E. coli JM 109 cells (Promega) using standardtechniques.

2. Cloning of a FEN-1 Endonuclease From Acidianus brierlyi

Cloning of the FEN-1 from Acidianus brierlyi (Abr) was performed asdescribed above, except the DSM#is 1651 and the PCR primers used are Abr5′-5′CATACCATGGGAGTAGATTTATCTGACTTAG (SEQ ID NO:338)and Abr3′-5′CTTGGTCGACTTAAAACCATTGGTCAAGTCCAG (SEQ ID NO:339). A ‘C’ atposition 3 of the Abr ORF sequence (SEQ ID NO:338) was mutated to a ‘G’in the 5′ primer to create an ATP start codon. The open reading frame(ORF) encoding the Abr FEN-1 endonuclease is provided in SEQ ID NO:340;the amino acid sequence encoded by this ORF is provided in SEQ IDNO:341.

3. Cloning of a FEN-1 Endonuclease From Aeropyrum pernix

Cloning of the FEN-1 from Aeropyrum pemix (Ape) was performed asdescribed above, except the sequence of this enzyme was obtained fromGENBANK (accession #H₇₂₇₆₅), the DSM#is 11879, and the PCR primers usedare Ape 5′ -5′TTAGCCATGGGAGTCAACCTTAGGGAG (SEQ ID NO:342) and Ape 3′-5′GTAAGTCGACTATCCGAACCACATGTCGAG (SEQ ID NO:343). An ‘T’ at position 1 ofthe Ape ORF sequence (SEQ ID NO:344) was mutated to an ‘A’ in the 5′primer to create an ATP start codon. The open reading frame (ORF)encoding the Ape FEN-1 endonuclease is provided in SEQ ID NO:344; theamino acid sequence encoded by this ORF is provided in SEQ ID NO:345.

4. Cloning of a FEN-1 Endonuclease From Archaeaglobus profundus

Cloning and expression of the FEN-1 from Archaeaglobus profundus (Apr)was performed as described above, except the DSM#is 5631 and the PCRprimers used are Apr 5′-5′CTTACCATGGGCGCTGATATAGGAGAGC (SEQ ID NO:346)and Apr 3′-5′TGGAGTCGACTTAAAACCACCTGTCCAGAG (SEQ ID NO:347). The openreading frame (ORF) encoding the Apr FEN-1 endonuclease is provided inSEQ ID NO:348; the amino acid sequence encoded by this ORF is providedin SEQ ID NO:349.

5. Cloning of a FEN-1 Endonuclease From Methanococcus igneus

Cloning and expression of the FEN-1 from Methanococcus igneus (Mig) wasperformed as described above except the DSM #is 5666 and the PCR primersused are Mig 5′-5′CATTCCATGGGAGTGCAGTTTAATG (SEQ ID NO:350) and Mig 3′-5′CGGAGTCGACTCATCTCCCAAACCATGC(SEQ ID NO:351). The open reading frame(ORF) encoding the Mig FEN-1 endonuclease is provided in SEQ ID NO:352;the amino acid sequence encoded by this ORF is provided in SEQ IDNO:353.

6. Cloning of a FEN-1 Endonuclease From Pyrococcus horikoshii

Cloning and expression of the FEN-1 from Pho Pyrococcus horikoshii (Pho)was performed as described above except the sequence of this enzyme wasobtained from GENBANK (accession #A71015), the DSM #is 12428, and thePCR primers used are Pho 5′-5′GATACCATGGGTGTTCCTATCGGTGAC (SEQ IDNO:354) Pho 3′-5′CTTGGTCGACTTAGGGTTTCTTTTTAACGAACC (SEQ ID NO:355). Theopen reading frame (ORF) encoding the Pho FEN-1 endonuclease is providedin SEQ ID NO:356; the amino acid sequence encoded by this ORF isprovided in SEQ ID NO:357.

7. Cloning of a FEN-1 Endonuclease From Sulfolobus solfataricus

Cloning and expression of Sulfolobus solfataricus (Sso) FEN-1 wasperformed as described above except the bacterial stocks were obtainedfrom the American Type Culture Collection (Manassas, Va.) ATCC #is 5091,and the PCR primers used are Sso 5′-5′TAAGCCATGGGTGTAGATTTAGGCGAAATAG(SEQ ID NO:358) Sso 3′-5′ACTAGTCGACTTAAAACCACTGATCAAGACCTGTC (SEQ IDNO:359). An ‘A’ at position 3 of the Sso ORF sequence (SEQ ID NO:360)was mutated to a ‘G’in the 5′ primer to create an ATP start codon. Theopen reading frame (ORF) encoding the Sso FEN-1 endonuclease is providedin SEQ ID NO:360; the amino acid sequence encoded by this ORF isprovided in SEQ ID NO:361.

8. Cloning of a FEN-1 Endonuclease From Thermococcus gorgonarius

Cloning and expression of Thermococcus gorgonarius (Tgo) was performedas above except the ATCC #is 700653D, and the PCR primers used are Tgo5′-5′CTAGCCATGGGAGTTCAGATAGGTGAGC (SEQ ID NO:362) and Tgo3′-5′TGGAGTCGACTACCGTGTGAACCAGCTTTC (SEQ ID NO:363). The open readingframe (ORF) encoding the Tgo FEN-1 endonuclease is provided in SEQ IDNO:364; the amino acid sequence encoded by this ORF is provided in SEQID NO:365.

II. Cloning of FEN-1 Endonucleases With EcoRI/SalI

The FEN-1's in this group were cloned as described above, except therestriction endonucleases used for the cloning step are EcoRI and SalI.This is due to the presence of an internal NcoI site in these FEN-1sequences. EcoRI is a good choice since it is common in the art ofcloning, is found in many expression vectors, and the following FEN-1'scontain no internal EcoRI sites. Interestingly, cloning these PCRfragments into the pTRC99a vector yields an ORF containing two aminoacids not present in the native sequence. To correct this and obtain anative ORF, mutagenesis reactions were performed with the Transformer™Site-Directed Mutagenesis Kit (Palo Alto Calif., cat #K1600-1) accordingto the manufacturer's instructions, using the mutagenic oligonucleotidespecified for each FEN-1 endonuclease. After the mutagenesis reaction,selection was achieved by cutting the products with EcoRI. Since themutant constructs have no EcoRI site, they will not be cut. Anyconstructs that were not properly mutagenized would still contain andEcoRI restriction site and would be cut during the digest reaction.Bacterial cells were then transformed and grown on selective medium(ampicillin) according to the Transformer™ Site-Directed Mutagenesis Kitinstructions. Mutangenic plasmid was isolated according to themanufacturer's instructions, the resultant DNA was again cut with EcoRI,and the products of this reaction were used to transform competent E.coli JM 109 cells (Promega) using standard techniques.

9. Cloning of a FEN-1 Endonuclease From Archaeaglobus veneficus

The cloning of a FEN-1 from Archaeaglobus veneficus (Ave) was performedas described above except the DSM #11195, the PCR primers used are Ave5′-5′TAACGAATTCGGTGCAGACATAGGCGAACTAC (SEQ ID NO:366) and Ave3′-5′CGGTGTCGACTCAGGAAAACCACCTCTCAAGCG (SEQ ID NO:367), and themutagenic oligonucleotide used was AveΔRI—5′CACAGGAAACAGACCATGGGTGCAGACATAGGCGAAC (SEQ ID NO:368). The openreading frame (ORF) encoding the Ave FEN-1 endonuclease is provided inSEQ ID NO:369; the amino acid sequence encoded by this ORF is providedin SEQ ID NO:370.

10. Cloning of a FEN-1 Endonuclease From Desulfurococcus amylolyticus

The cloning of a FEN-1 from Desulfurococcus amylolyticus (Dam) wasperformed as described above except the DSM #3822, the PCR primers usedare Dam 5′-5′CTAAGAATTCGGAGTAGACTTAAAAGACATTATACC (SEQ ID NO:371) andDam 3′-5′AGTTGTCGACTACTTCGGCTTACTGAACC (SEQ ID NO:372), and themutagenic oligonucleotide used was DamΔRI—5′CAGGAAACAGACCATGGGAGTAGACTTAAAAGAC (SEQ ID NO:373). The openreading frame (ORF) encoding the Dam FEN-1 endonuclease is provided inSEQ ID NO:374; the amino acid sequence encoded by this ORF is providedin SEQ ID NO:375.

11. Cloning of a FEN-1 Endonuclease From Pyrobaculum aerophilum

The cloning of a FEN-1 from Pyrobaculum aerophilum (Pae)was performed asdescribed above except the DSM #7523, the PCR primers used were Clonedusing EcoR I and Sal I Pae 5′-5′CGTTGAATTCGGAGTTACTGAGTTGGGTAAG (SEQ IDNO:376) and Pae 3′-5′TACTGTCGACAGAAAAAGGAGTCGAGAGAGGAAG (SEQ ID NO:377).A‘G’ at position I of the Pae ORF sequence (SEQ ID NO:378) was mutatedto an ‘A’ in the 5′ primer to create an ATP start codon. The mutagenesisreaction for this enzyme has not yet been done. There are two,non-native amino acids at the 5′ end of this protein. The open readingframe (ORF) encoding the Pae FEN-1 endonuclease is provided in SEQ IDN0378; the amino acid sequence encoded by this ORF is provided in SEQ IDNO:379.

12. Cloning of a FEN-1 Endonuclease From Thermococcus litoralis

The cloning of a FEN-1 from Thermococcus litoralis (Tli) was performedas described above except the DSM#is 5473, the PCR primers used were Tli5′-5′TCATGAATTCGGAGTCCAGATTGGTGAGCTT (SEQ ID NO:380) and Tli3′-5′GATTGTCGACTCACTTTTTAAACCAGCTGTCC (SEQ ID NO:381), and the mutagenicprimer was Tli ΔRI-5′AAGCTCACCAATCTGGACTCCCATGGTCTGTTTCCTGTG (SEQ IDNO:382). The open reading frame (ORF) encoding the Tli FEN-1endonuclease is provided in SEQ ID NO:383; the amino acid sequenceencoded by this ORF is provided in SEQ ID NO:384.

13. Cloning of a FEN-1 Endonuclease From Desulfurococcus mobilis

The cloning of a FEN-1 from Desulfurococcus mobilis (Dmo) was performedas described above except the DSM #2161, the PCR primers used were Dmo5′-5′CTTGGAATTCGGCGTCGACCTAAGGGAACTC (SEQ ID NO:385) and Dmo3′-5′AGGTCTGCAGTTAACCCTGCTTACCGGGCTTAGC (SEQ ID NO:386), and themutagenic primer used was Dmo ΔR1-5′CAGGAAACAGACCATGGGCGTCGACCTAAGG (SEQID NO:387). The open reading frame (ORF) encoding the Dmo FEN-1endonuclease is provided in SEQ ID NO:388; the amino acid sequenceencoded by this ORF is provided in SEQ ID NO:389.

14. Cloning of a FEN-1 Endonuclease From Pyrodictium brockii

The cloning of a FEN-1 from Pyrodictium brockii (Pbr) was performed asdescribed above, except the DSM #2708, the PCR primers used were Pbr5′-5′TAGCGAATTCGGCGTCAACCTCCGCGAG (SEQ ID NO:390) and Pbr3′-5′CATTCTGCAGCTAGCGGCGCAGCCACGC (SEQ ID NO:391), and the mutagenicprimer was Pbr ΔR1-5′CAGGAAACAGACCATGGGCGTCAACCTCCGC (SEQ ID NO:392). A‘G’ at position 1 of the Pbr ORF sequence (SEQ ID NO:393) was mutated toan ‘A’ in the 5′ primer to create an ATP start codon. The open readingframe (ORF) encoding the Pbr FEN-1 endonuclease is provided in SEQ IDNO:393; the amino acid sequence encoded by this ORF is provided in SEQID NO:394.

III. Cloning of FEN-1 Endonucleases With Other Enzymes

The following FEN-1 endonucleases were cloned using restrictionendonucleases other than those already described. These particular FEN-1sequences contain either internal EcoRI sites or internal SalI sites andtherefore require different restriction enzymes in the cloning step.None of the PCR primers used in this section yield cloned productscontaining an ORF with aberrant (non-natural) nucleotides, thereforethere is no need for an additional mutagenesis step. All reactionsdescribed in this section were performed as in section I, with anyexceptions noted below.

15. Cloning of a FEN-1 Endonuclease From Methanopyrus kandleri

The cloning of a FEN-1 from Methanopyrus kandleri (Mka) was performed asdescribed above, except the DSM #6324 and the PCR primers used were Mka5′-5′CATACCATGGGACTAGCTGAACTCCGAG (SEQ ID NO:395) and Mka3′-5′TGGATCTAGATCAGAAGAACGCGTCCAGGG (SEQ ID NO:396). A ‘T’ at position 1of the Mka ORF sequence (SEQ ID NO:397) was mutated to an ‘A’ in the 5′primer to create an ATP start codon. The restriction enzymes used in thecloning reaction were NcoI and XbaI. The open reading frame (ORF)encoding the Mka FEN-1 endonuclease is provided in SEQ ID NO:397; theamino acid sequence encoded by this ORF is provided in SEQ ID NO:398.

16. Cloning of a FEN-1 Endonuclease From Thermococcus zilligii

The cloning of a FEN-1 from Thermococcus zilligii (Tzi) was performed asdescribed above, except genomic DNA obtained was obtained from theAmerican Type Culture Collection (ATCC #700529D) and the PCR primersused were Tzi 5′-5′CGATCCATGGGAGTTCAGATCGGTGAGC (SEQ ID NO:399) and Tzi3′-5′CAGGCTGCAGTCACCTTCCGAACCAGCTCTC (SEQ ID NO:400). The restrictionenzyme used in the cloning reaction were NcoI and PstI. The open readingframe (ORF) encoding the Tzi FEN-1 endonuclease is provided in SEQ IDNO:401; the amino acid sequence encoded by this ORF is provided in SEQID NO:402.

IV. Site Directed and Recombinant Mutants of FEN1 Enzymes.

Arch Swap Chimeras

The arch region of the FEN1 enzymes comprises approximately 58 aminoacids from amino acid 78 (beginning with the conserved VFDG sequence) toamino acid 135. The FEN1 enzymes from Archaeoglobus fulgidus (Afa),Pyrococcus furiosus (Pfu), and Methanococcus jannaschii (Mja) havedifferent specificities, the arch regions were ‘swapped’ between theenzymes and ethe effects on enzyme specificity were examined. Aminoacids 78 through 84 (beginning of the arch region) are identical in theAfu, Pfu and Mja FEN1 enzymes, amino acids 85 to 135 of the Mja, Pfu andAfu were swapped to create new chimeric enzymes. The four enzymescreated are MPM, PMP, MAM, and PAP.

MPM consists of amino acids 1-84 of the Mja FEN1 enzyme, amino acids85-135 of the Pfu FEN1 enzyme, and amino acids 136-326 of the Mja FEN1enzyme. PMP consists of amino acids 1-84 from the Pfu FEN1 enzyme, aminoacids 85-135 of the Mja FEN1 enzyme, and amino acids 136-340 of the PfuFEN1 enzyme. MAM consists of amino acids 1-84 of the Mja FEN1 enzyme,amino acids 85-134 of the Afu FEN1 enzyme, and amino acids 136-326 ofthe Mja FEN1 enzyme (the numbering discrepancy is due to the fact thatthe arch is one amino acid shorter in Afu FEN1 than in Pfu and MjaFENIs). PAP consists of amino acids 1-84 of the Pfu FEN1 enzyme, aminoacids 85-134 of the Afu FEN1 enzyme, and amino acids 136-340 of the PfuFEN1 enzyme.

The MPM chimera was constructed in the following way. Three PCRreactions were set up, one for each fragment in the chimera. PCRreactions were carried out as described. Reaction 1 used primers Mja5′(5′-GGGATACCATGGGAGTGCAGTTTGG, SEQ ID NO:411) and MPM84AS 5′-5(GAGCTCTTTCTTTTTGAATTCTGGTGGCTCACCATCAAAAAC, SEQ ID NO:412) and 1 ng ofa Mja FEN1 gene containing plasmid. Reaction 2 used primers P84S(5′-GAATTCAAAAAGAAAGAGCTC, SEQ ID NO:413) and P135AS(5′-TGCATCCTCGATGAGCATTTC, SEQ ID NO:414) and 1 ng of a Pfu FEN1 genecontaining plasmid. Reaction 3 used primers MPM135S(5′-GAAATGCTCATCGAGGATGCAAAATATTTGTTAAGTTTGATGGG, SEQ ID NO:415) andMja3′ (5′-GGTAAATTTTTCTCGTCGACATCCCAC, SEQ ID NO:416) and 1 ng of a MjaFEN1 gene containing plasmid. After the first reaction, the fragmentswere gel purified and eluted into 50 μl of elution buffer. The DNAfragments were diluted 1 to 10 in water and 1 μl of each dilutedfragment was added to the final PCR which was done as described with theMja5′ and Mja3′ primers. After the final PCR, the DNA was gel purifiedand cloned into the pTrc99a vector as described. The MPM open readingframe sequence is SEQ ID NO 417; the MPM amino acid sequence is SEQ IDNO:418.

The PMP chimera was constructed in the following way. Three PCRreactions were set up, one for each fragment in the chimera. PCRreactions were carried out as described. Reaction 1 used primers Pfu5′(5′-GATTCCATGGGTGTCCCAATTGGTGAG, SEQ ID NO:419) and Pmp84AS(5′-CCTTGTTTTCTCCTTTAACTTTGGAGGTTCTCCATCAAAAAC, SEQ ID NO:420) and 1 ngof a Pfu FEN1 gene containing plasmid. Reaction 2 used primers M84S(5′-AAGTTAAAGGAGAAAACAAGG, SEQ ID NO:421) and M135AS(5′-GCAGTTTTCAACCATTTTCGG, SEQ ID NO:422) and 1 ng of a Mja FEN1 genecontaining plasmid. Reaction 3 used primers Pmpl35S(5′-CCGAAAATGGTTGAAAACTGCAAAAAACTCTTAGAGCTTATG, SEQ ID NO:423) and Pfu3′(5′-CGGAGTCGACTTATCTCTTGAACCAACTTTCAAG, SEQ ID NO:424) and 1 ng of a PfuFEN1 gene containing plasmid. After the first reaction, the fragmentswere gel purified and eluted into 50 μl of elution buffer. The DNAfragments were diluted 1 to 10 in water and 1 μl of each dilutedfragment was added to the final PCR which was done as described with thePfu5′ and Pfu3′ primers. After the final PCR, the DNA was gel purifiedand cloned into the pTrc99a vector as described. The PMP open readingframe sequence is SEQ ID NO:425, the PMP amino acid sequence is SEQ IDNO:426.

The MAM chimera was constructed in the following way. Three PCRreactions were set up, one for each fragment in the chimera. PCRreactions were carried out as described. Reaction 1 used primers Mja5′and MAM84AS (5′-CAATTTCAGCCTTCTTGAACTCTGGTGGCTCACCATCAAAAAC, SEQ IDNO:427) and 1 ng of a Mja FEN1 gene containing plasmid. Reaction 2 usedprimers A84S (5′-GAGTTCAAGAAGGCTGAAATTG, SEQ ID NO:428) and A135AS(5′-TGCGGAGTCAACAATGTACTC, SEQ ID NO:429) and 1 ng of a Afu FEN1 genecontaining plasmid. Reaction 3 used primers MAM135S(5′-GAGTACATTGTTGACTCCGCAAAATATTTGTTAAGTTTGATGGGC, SEQ ID NO:430) andMja3′ and 1 ng of a Mja FEN1 gene containing plasmid. After the firstreaction, the fragments were gel purified and eluted into 50 μl ofelution buffer. The DNA fragments were diluted 1 to 10 in water and 1 μlof each diluted fragment was added to the final PCR which was done asdescribed with the Pfu5′ and Pfu3′ primers. After the final PCR, the DNAwas gel purified and cloned into the pTrc99a vector as described. TheMAM open reading frame sequence is SEQ ID NO:431; the MAM amino acidsequence is SEQ ID NO:432.

The PAP chimera was constructed in the following way. Three PCRreactions were set up, one for each fragment in the chimera. PCRreactions were carried out as described. Reaction 1 used primers Pfu5′and PAP84AS (5′-CAATTTCAGCCTTCTTGAACTCTGGAGGTTCTCCATCAAAAAC, SEQ IDNO:433) and 1 ng of a Pfu FEN1 gene containing plasmid. Reaction 2 usedprimers A84S and A135AS and 1 ng of a Afu FEN1 gene containing plasmid.Reaction 3 used primers PAP135S(5′-GAGTACATTGTTGACTCCGCAAAAAAACTCTTAGAGCTTATGGG, SEQ ID NO:434) andPfu3′ and 1 ng of a Pfu FEN1 gene containing plasmid. After the firstreaction, the fragments were gel purified and eluted into 50 μl ofelution buffer. The DNA fragments were diluted 1 to 10 in water and 1 ulof each diluted fragment was added to the final PCR which was done asdescribed with the Pfu5′ and Pfu3′ primers. After the final PCR, the DNAwas gel purified and cloned into the pTrc99a vector as described. ThePAP open reading frame sequence is SEQ ID NO:435; the PAP amino acidsequence is SEQ ID NO 436.

Loop Swap Chimeras

When the sequence of the FEN1 enzymes are compared, most of the ArchaealFEN1 enzymes, with the exception of Mja FEN1 and Mth FEN1, have 10additional amino acids, when compared to the eukaryotic FEN1 enzymes.This amino acid sequence forms a Bribbon loop that protrudes from thesurface of the enzyme. Since both Mja FEN1 and Mth FEN1 have highactivity on hairpin and X-structure substrates and they lack this loopsequence, tests were conducted to determine the effect of the presenceof the loop on enzyme specificity.

Two chimeric enzymes were constructed: Mja+Pfu16, which is the Mja FEN1enzyme with the Pfu loop sequence inserted into it at the properlocation; and Pfu+Mja4, which is the Pfu enzyme with the loop sequencedeleted and 4 amino acids from Mja FEN1 inserted in its place. Bothenzymes were created by recombinant PCR.

For the creation of Mja+Pfu16, two PCR reactions were performed. Thefirst reaction contained Mja5′ and Mja+Pfu16AS(5′-CGTAGACATTTTTCCCAGGCAACTTTCTTTTTCCTGTAGTTGTTAAATTTCTAA C, SEQ IDNO:437) primers and 1 ng of Mja FEN1 containing plasmid. The secondreaction contained Mja+Pfu16S(5′-CCTGGGAAAAATGTCTACGTCGAGATAAAGCCCGAACTTATTGAATTAAATG, SEQ ID NO:438)and Mja3′ primers and 1 ng of MjaFEN1 containing plasmid. After thefirst reaction, the fragments were gel purified and eluted into 50 μl ofelution buffer. The DNA fragments were diluted 1 to 10 in water, and 1μl of each diluted fragment was added to the final PCR, which was doneas described with the Mja5′ and Mja3′ primers. After the final PCR, theDNA was gel purified and cloned into the pTrc99a vector as described.The Mja+Pfu 16 open reading frame sequence is SEQ ID NO:439; theMja+Pfu16 amino acid sequence is SEQ ID NO:440.

For the creation of Pfu+Mja4, two PCR reactions were set up. The firstreaction contained Pfu5′ and Pfu+Mja4AS(5′-CTCTGGCATCTCCTTTGTTATTGTTAAGTTTCTAAC, SEQ ID NO:441) primers and 1ng of Pfu FEN1 containing plasmid. The second reaction containedPfu+Mja4S (5′-ACAAAGGAGATGCCAGAGTTGATAATTTTGGAGGAAG, SEQ ID NO:442) andPfu3′ primers and 1 ng of PfuFEN1 containing plasmid. After the firstreaction, the fragments were gel purified and eluted into 50 μl ofelution buffer. The DNA fragments were diluted 1 to 10 in water and 1 μlof each diluted fragment was added to the final PCR which was done asdescribed with the Pfu5′ and Pfu3′ primers. After the final PCR, the DNAwas gel purified and cloned into the pTrc99a vector as described.Enzymes were purified as described. The Pfu+Mja4 open reading framesequence is SEQ ID NO:443; the Pfu+Mja4 amino acid sequence is SEQ IDNO:444.

Mutations in Conserved Tyrosine

In modeling studies of the FEN1 enzymes with DNA substrates, it wasobserved that the 3′ end of the INVADER oligonucleotide is in closeproximity to a conserved tyrosine at position 33 of the Pfu FEN1 proteinsequence. That amino acid is a tyrosine in all of the known ArchaealFEN1 sequences, and is phenylalanine or tyrosine in the bacterial DNApolymerase I homologues. The tyrosine at position 33 of the Pfu FEN1enzyme was mutated to an alanine or a phenyalanine to determine itseffect on recognition of the 3′ end of the INVADER oligonucleotide. Themutations were made individually by site directed mutagenesis using theQuikChange technique (Stratagene). The oligonucleotides used in the Y33Amutagenesis were PfaY33A-S (5′-CTCTTAATGCAATCGCCCAATTTTTGTCCAC, SEQ IDNO:445) and Pfu Y33A-AS (5′- GTGGACAAAAATTGGGCGATTGCATTAAGAG, SEQ IDNO:446). The oligonucleotides used in the Y33F mutagenesis werePfuY33F-S (5′-CTCTTAATGCAATCTTCCAATTTTTGTCCAC, SEQ ID NO:447) andPfuY33F-AS (5′-GTGGACAAAAATTGGAAGATTGCATTAAGAG, SEQ ID NO:448). ThePfuY33F open reading frame sequence is SEQ ID NO:449; the PfuY33F aminoacid sequence is SEQ ID NO:450. The PfuY33A open reading frame sequenceis SEQ ID NO:451; the PfuY33A amino acid sequence is SEQ ID NO:452.Following mutagenesis and screening for the presence of the mutations,enzymes were purified as described.

Pho and Pfu and Arch Mutants

The Pho FEN1 enzyme has a cleavage rate on hairpin and X-structuresubstrates that is 1 to 2 orders of magnitude greater than the samerates measured using the Pfu FEN1 enzyme. When the amino acid sequencesof the two enzymes were compared, we noted that there were 4 changesbetween the two enzymes in the arch region (at amino acid positions 82,103, 110, and 113). At each of the positions where differences occurbetween the two enzymes, Pfu FEN1 has a negatively charged glutamic acidresidue while Pho FEN1 has a neutral or positively charged amino acid atthose positions. Tests were conducted to examine the effects of theseresidues on the sepecificities of these enzymes. We made mutants of thePfu and Pho FEN1 enzymes to swap the amino acids in the arch region anddetermine the mutations' effects on specificity. The followingconstructs were made using the QuikChange mutagenesis technique(Stratagene). For constructs with multiple mutations, sequentialmutagenesis reactions were done. Pfu1M has the E82K mutation made usingoligonucleotides PfuE82K-S (5′-GTGTATGTTTTTGATGGAAAACCTCCAGAATTC, SEQ IDNO:453) and PfuE82K-AS (5′-GAATTCTGGAGGTTTTCCATCAAAAACATACAC, SEQ IDNO:454). Pfu2M has the E82K mutation and the E103L mutation made usingoligonucleotides PfuE103L-S (5′-GAGAGAGGAAGCTGAACTAAAGTGGAGAGAAGC, SEQID NO:455) and PfuE103L-AS (5′-GCTTCTCTCCACTTTAGTTCAGCTTCCTCTCTC, SEQ IDNO:456). Pfu3M has the E82K and E103L mutations, and the E110A mutationmade with PfuE110A-S (5′-GAGAGAAGCACTTGCAAAAGGAGAGATAGAGG, SEQ IDNO:457) and PfuE110A-AS (5′-CCTCTATCTCTCCTTTTGCAAGTGCTTCTCTC, SEQ IDNO:458). Pfu4M has the E82K, E103L and E110A mutations and the E113Nmutation made with PfuE113N-S (5′-GAAGCACTTGCAAAAGGAAACATAGAGGAAGCAAGAA,SEQ ID NO:459) and PfuE 113N-AS(5′-TTCTTGCTTCCTCTATGTTTCCTTTTGCAAGTGCTTC, SEQ ID NO:460). Pho1M has theK82E mutation made with PhoK82E-S(5′-CCTACGTCTTTGATGGAGAGCCTCCGGAATTCAAAAGG, SEQ ID NO:461) andPhoK82E-AS (5′-CCTTTTGAATTCCGGAGGCTCTCCATCAAAGACG, SEQ ID NO:462). Pho2Mhas the K82E mutation and the L103E mutation made with PhoL103E-S(5′-GAGAAGAGGCAGAAGAAAAATGGAAAGAAGC, SEQ ID NO:463) and PhoL103E-AS(5′-GCTTCTTTCCATTTTTCTTCTGCCTCTTCTC, SEQ ID NO:464). Pho3M has the K82Eand L103E mutations and the A110E mutation made with PhoA110E-S(5′-GGAAAGAAGCTCCAGAGAAGGGAAACCTGGAGG, SEQ ID NO:465) and PhoA110E-AS(5′-CCTCCAGGTTTCCCTTCTCTAGAGCTTCTTTCC, SEQ ID NO:466). Pho4M has theK82E, L103E and A110E mutations and the N113E mutation made withPhoN113E-S (5′-GCTCTAGAGAAGGGAGAACTGGAGGAAGCTAGG, SEQ ID NO:467) andPhoN 113E-AS (5′-CCTAGCTTCCTCCAGTTCTCCCTTCTCTAGAGC, SEQ ID NO:468).

The Pfu1M open reading frame sequence is SEQ ID NO:469; the Pfu1M aminoacid sequence is SEQ ID NO:470. The Pfu2M open reading frame sequence isSEQ ID NO:471; the Pfu2M amino acid sequence is SEQ ID NO:472. The Pfu3Mopen reading frame sequence is SEQ ID NO:473; the Pfu3M amino acidsequence is SEQ ID NO:474. The Pfu4M open reading frame sequence is SEQID NO:475; the Pfu4M amino acid sequence is SEQ ID NO:476. The Pho1Mopen reading frame sequence is SEQ ID NO:477; the Pho1M amino acidsequence is SEQ ID NO:478. The Pho2M open reading frame sequence is SEQID NO:479; the Pho2M amino acid sequence is SEQ ID NO:480. The Pho3Mopen reading frame sequence is SEQ ID NO:481; the Pho3M amino acidsequence is SEQ ID NO:482. The Pho4M open reading frame sequence is SEQID NO:483; the Pho4M amino acid sequence is SEQ ID NO:484.

Mutation Affecting 3′ End Interaction

The mutations in position Y33 of Pfu FEN1 were made because it wasbelieved that amino acid was interacting with the 3′ end of the INVADERoligonucleotide. To test that hypothesis, Pfu, PfuY33F, and PfuY33A FEN1enzymes were tested on INVADER oligonucleotides with different 3′ endsto determine the effect of the mutations on cleavage of thosesubstrates. The combined target-probe oligonucleotide 203-15-2(5′-TET-AAAACGCTGTCTCGCTGAAAGCGAGACAGCGAAAGACGCTCGT, SEQ ID NO:485) wasused with INVADER oligonucleotides that differ only at their 3′ end. Thedifferent INVADER oligonucleotides are 203-15-3 (5′-ACGAGCGTCTTT, SEQ IDNO:486), 203-15-4 (5′-ACGAGCGTCTTTA, SEQ ID NO:487), 203-15-5(5′-ACGAGCGTCTTTT, SEQ ID NO:488), 203-15-6 (5′-ACGAGCGTCTTTG, SEQ IDNO:489), 203-15-7 (5′-ACGAGCGTCTTTC, SEQ ID NO 490), 203-15-8(5′-ACGAGCGTCTTT-dideoxyC, SEQ ID NO:491), 203-15-9(5′-ACGAGCGTCTTTC-PO₄, SEQ ID NO:492), and 203-15-10(5′-ACGAGCGTCTTT-dribose, SEQ ID NO:493). 10 μl reactions contained 2 μM203-15-2, and 2 μM of one of INVADER oligonucleotides (above), 10 mMMOPS, pH 7.5, 0.05% TWEEN 20, 0.05% NP-40, 4 mM MgCl2, and 0.35 nM (PfuFEN1 and PfuY33F FEN1) or 3.5 nM (PfuY33A FEN1) enzyme. Reactions wererun at 50 C for between 10 to 30 minutes. Reactions were stopped by theaddition of 10 μl of 95% formamide, 0.02% methyl violet. 1 μl of themixture was run on a denaturing 20% acrylamide gel and electrophoresedfor 15 minutes or until good separation was achieved between cleaved anduncleaved probe. The gels were scanned using an FMBIO fluorescent gelscanner and the bands were quantified with FMBIOAnalysis software.Cleavage rates were determined from the percent probe cleaved. Thecleavage rate is defined as the number of probe-target oligonucleotidescleaved per minute per enzyme. Though the cleavage rate is reduced forPfu Y33F and more so for Pfu Y33A, no change in specificity is observed.The cleavage rate versus the 3′ end is plotted for the 3 enzymes (FIG.148).

Example 66 Activity Assay for Cloned FEN1 Nucleases

The INVADER assay test was done using a combination target and upstream(INVADER) oligonucleotide and a large molar excess of labeled probeoligonucleotide (as diagrammed FIG. 141A). Ten μl reactions contained 10mM MOPS (pH 7.5), 0.05% NP-40, 0.05% Tween 20, 20 ng/μl tRNA (Sigma), 4mM MgCl2, 100 ng enzyme, 2 μM labeled probe 203-91-01(5′-(Tet)TTTTCAACTGCCGTGA; SEQ ID NO:403) and 0.2 nM target-INVADER203-91-04 (5′-TCACGGCAGTTGGTGCGCCTCGGAACGAGGCGCACA; SEQ ID NO:404),.Reactions were set up by adding all components to individual wells of a96-well plate in an ice water bath. The reactions were started byplacing the 96-well plate on a prewarmed gradient thermal cycler(Eppendorf Mastercycler) and incubated at the set temperature for 20minutes. The 96-well plate was then put back in the ice water bath tostop the reactions and 10 μl of stop mix (95% formamide, 20 mM EDTA,0.05% methyl violet) was added. 1 μl of each reaction was loaded onto adenaturing 20% acrylamide gel. After electrophoresis, the gels werevisualized using an FMBIO fluoroimager (Hitachi) and quantitated withFMBIO Analysis software. Results are shown graphically in FIG. 142.

Cleavage of an ‘X-structure’ as diagrammed in FIG. 141B can provide onesource of background signal in the sequential invasive cleavagereactions of the present invention, thus it is desireable to determinethe level of activity that enzymes have in cleaving such a structure.Ten-μl reactions on the X-structure substrate contained 10 mM MOPS (pH7.5), 0.05% NP-40, 0.05% Tween 20, 20 ng/μl tRNA (Sigma), 4 mM MgCl2,100 ng enzyme, and 2 μM labeled 203-81-02(5′-(Tet)TTTTCAACTGCTTAGAGAATCTAAGCAGTTGGTGCGCCTCGTTAA-NH2; SEQ IDNO:405) and 2 μM target 594-09-01 (5′-AACGAGGCGCACATTTTTTTT; SEQ IDNO:406). Reactions were done as described above except incubation at thereaction temperature was carried out for 60 minutes. Samples wereelectrophoresed and analyzed as described above. Results are showngraphically in FIG. 143.

Together these test allow characterization of the cleavage activities ofFEN enzymes and other 5′ nucleases, and of any new enzyme suspected ofbeing able to cleave an invasive cleavage structure.

Evaluation of Chimeric and Mutant FEN1 Enzymes

For the evaluation of chimeric enzymes, standard tests were performed tomeasure the performance and specificity of the enzymes. The INVADER testassay contains 10 mM MOPS, pH 7.5, 0.05% TWEEN 20, 0.05% NP-40, 4 mMMgCl2, 0.5nM target-INVADER oligonucleotide 594-12-1(5′-Biotin-TTTTTCACGGCAGTTGGTGCGCCTCGGAACGAGGCGCACA, SEQ ID NO:517), 1μM probe oligonucleotide 203-91-1 (5′-Tet-TTTTCAACTGCCGTGA, SEQ IDNO:518), 2 nM streptavidin and 256 nM enzyme. 10 μl reactions are run at59 C for between 5 to 60 minutes depending on the enzyme. Reactions arestopped by the addition of 10 μl of 95% formamide, 0.02% methyl violet.1 μl of the mixture is run on a denaturing 20% acrylamide gel andelectrophoresed for 15 minutes or until good separation is achievedbetween cleaved and uncleaved probe. The gels are scanned using an FMBIOfluorescent gel scanner and the bands are quantified with FMBIOAnalysissoftware. Cleavage rates are determined from the percent probe cleaved.Cleavage rate for the INVADER substrate is defined as the number ofprobe molecules cleaved per target molecule per minute. In Table 5,below, the cleavage rates for the mutant enzymes are compared to thecleavage rates for unmodified Pfu FEN1 enzyme on the INVADER assay andX-structure substrates.

The X-structure substrate is used to determine the amount of backgroundcleavage an enzyme will carry out in an INVADER reaction. It mimics thestructure created by uncut primary probe and secondary FRET cassette inthe INVADER squared reaction. X-structure reactions contain 10 mM MOPS,pH 7.5, 0.05% TWEEN 20, 0.05% NP-40, 4 mM MgCl2, 2 μM target-probeoligonucleotide 203-81-2(5′-TET-TTTTCAACTGCTTAGAGAATCTAAGCAGTTGGTGCGCCTCGTTAA-NH2;SEQ IDNO:494), 2 μM probe oligonucleotide 203-91-2 (5′-AACGAGGCGCACATT; SEQ IDNO:495), and 4 to 256 nM enzyme. 10 μl reactions are run at 59 C forbetween 3 to 30 minutes depending on the enzyme. Reactions are stoppedby the addition of 10 μl of 95% formamide, 0.02% methyl violet. 1 μl ofthe mixture is run on a denaturing 20% acrylamide gel and electophoresedfor 15 minutes or until good separation is achieved between cleaved anduncleaved probe. The gels are scanned using an FMBIO fluorescent gelscanner and the bands are quantified with FMBIOAnalysis software.Cleavage rates are determined from the percent probe cleaved. Thecleavage rate for the X-structure substrate is defined as the number ofprobe-target molecules cleaved per minute per molecule of enzyme.

TABLE 5 Rates of cleavage of modified FEN1 enzymes on INVADER and X-structure substrates (relative to the rates for unmodified Pfu FEN1).Relative INVADER Relative X-structure Enzyme Assay Cleavage rate^(a)cleavage rate^(b) Pfu FEN1 1 1.0 Pho FEN1 1.6 120 Pfu1M FEN1 0.83 0.85Pfu2M FEN1 1.4 3.0 Pfu3M FEN1 1.8 4.9 Pfu4M FEN1 2.2 62 Pho1M FEN1 1.118 Pho2M FEN1 1.6 31 Pho3M FEN1 0.95 6.6 Pho4M FEN1 1.3 2.2 Mja FEN1 1.9203 Afu FEN1 0.52 0.50 MPM FEN1 0.14 0.10 PMP FEN1 2.4 1.37 MAM FEN10.03 0.07 PAP FEN1 0.56 0.52 Pfu + M4 FEN1 0.05 0.28 Mja + P16 FEN1 1.259 Pfu Y33F FEN1 0.96 N.D. Pfu Y33A FEN1 0.05 N.D. ^(a)The INVADER assaycleavage rate is relative to the cleavage rate of the Pfu FEN1 enzymewhich has been given a value of 1. ^(b)The X-structure cleavage rate isrelative to the cleavage rate of the Pfu FEN1 enzyme which has beengiven a value of 1. N.D.—Not determined.

Example 67 High-throughput Screening for Selection Candidate Enzymes

For high-throughput screening, individual colonies are picked andinoculated into 1 ml of Luria-Bertani (LB) broth supplemented with 100μg/ml ampicillin and 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) indeep-well 96 well plates. Cultures are grown for approximately 20 hourswith vigorous shaking at 37 C. The culture plates are then put on theautomated robotic setup (e.g., as depicted in FIG. 147) for screening.

The assay plates are set up as follows. For each overnight culture, 4assay wells are created, 2 containing no target and 2 containing target(described below). In this way, each 96 well plate of overnight culturesgenerates one 96 well plate of lysates and one 384 well plate of assays.The lysates can be screened under additional conditions, e.g., in thepresence of salt, or denaturants such as guanidine hydrochloride, bothof which are known to inhibit the cleavage enzymes of the presentinvention. In such a case, each 96 well plate of lysates could yield 2or more assay plates.

The following steps are generally done robotically in a 96 well (celllysis) or 384 well (activity assay) format (e.g., as diagrammed in FIG.147).

Cell Lysis

75 μl of overnight culture is mixed with 100 μl of 0.02% TWEEN 20, 1mg/ml lysozyme and incubated at room temperature for 15 min. The lysisplates are then heated at 83 C for 3 minutes and the plates are spun at1100×G for 5 minutes at room temperature to pellet cellular debris.

Assay on DNA Targets

Plasmids containing the Factor II mutant and Factor V wild type targetswere created for use in the high throughput assays. The Factor II mutanttarget plasmid contains a 345 base pair insert from a mutant Factor II(Human prothrombin gene, SEQ ID NO:510) inserted into pCR-Topo2.1(Invitrogen). The Factor V wild type target plasmid contains a 220 basepair insert (SEQ ID NO:509) from the wild type human Factor V geneinserted into pCR-Topo2.1 (Invitrogen). For assaying cell lysates, 5 μlof cell lysate are added to 10 μl of buffer, probe oligonucleotides,INVADER oligonucleotides, FRET oligonucleotides and target mix. Thefinal concentration of the buffer components is 3.5% PEG, 10 mM MOPS, pH7.5, 14 mM MgCl2. 15 μl reactions contain 0.33 μM each of Factor R Fretprobe (5′-FI-TCT-Z28-AGCCGGTTTTCCGGCTGAGACGTCCGTGGCCT-Hex, SEQ IDNO:516) and Factor V Fret probe(5′-Red-TCT-Z28-AGCCGGTTTTCCGGCTGAGACGGCCTCGCG-Hex, SEQ ID NO:513), 0.66μM each of Factor V wild type probe 23-445(5′-CGCGAGGCCGGAGGAATACAGGTATTTTGTCC-Hex, SEQ ID NO:511) and Factor IImutant probe 23-215 (5′-AGGCCACGGACGAAGCCTCAATGCTCC-Hex, SEQ ID NO:514),and 66 nM Factor V INVADER oligonucleotide 23-200(5′-TCTAATCTGTAAGAGCAGATCCCTGGACAGACC, SEQ ID NO:512) and Factor IIINVADER oligonucleotide 23-415 (5′-TATGGTTCCCAATAAAAGTGACTCTCAGCT, SEQID NO:515). Between 20 and 100 pg of each plasmid target (describedabove) is used per reaction.

After mixing of the reagents, the 384 well plates are incubated at 63 Cfor 1 hour, after which the signal is read in a CYTOFLUOR fluorescenceplate reader. The wave length settings are 485 nm excitation and 530 nmemission for the fluorescein-containing probe and 560 nm excitation and620 nm emission for the REDMOND RED-dye containing probe. Fluorescencemeasurements are compared to measurements made using unmodified enzymesin the same reaction conditions. Enzymes showing increased signal in aparticular set of reaction conditions (i.e., more fluorescence) areconsidered for additional analysis. Generally, mutant enzymes showingincreases of at least 20 to 50% are further purified for for additionaltesting.

Assay on RNA Targets:

For testing activities of enzymes on RNA targets, INVADER assay probessets for the detection of Human ubiquitin mRNA and for Human Interleukin8 (IL-8) are used. For assaying cell lysates, 5 μl of cell lysate areadded to 10 μl of buffer, probe oligonucleotides, INVADERoligonucleotides, FRET oligonucleotides and target mix. The finalconcentration of the buffer components in the primary reaction are 4%PEG, 12.5 mM MgSO₄, 10 mM MOPS pH 7.5, 100 mM KCl, 0.05% Tween 20 and0.05% NP-40. The Primary reactions are incubated at 60° C. for 1 hour.The secondary reactions are then started by the addition of thesecondary reaction mix comprising the arrestor oligonucleotides and thesecondary probe and-target oligonucleotide sets. The finalconcentrations of the buffer components in the Secondary reaction are2.67% PEG, 20 mM MgSO₄, 6.67 mM MOPS pH 7.5, 66.7 mM KCl, 0.033% Tween20, and 0.033% NP-40. Secondary reactions are incubated at 60° C. for 1hour, after which the signal is read in a CYTOFLUOR fluorescence platereader. The wavelength settings are 485 nm excitation and 530 nmemission for the fluorescein-containing probe and 560 nm excitation and620 nm emission for the REDMOND RED-dye containing probe. Fluorescencemeasurements are compared to measurements made using unmodified enzymesin the same reaction conditions. Enzymes showing increased signal in aparticular set of reaction conditions (i.e., more fluorescence) areconsidered for additional analysis. Generally, mutant enzymes showingincreases of at least 20 to 50% are further purified for for additionaltesting.

Primary reactions comprise Hu ubiquitin INVADER oligonucleotide (0.5 μM,5′CCTTCCTTATCCTGGATCTTGGCA, SEQ ID NO:496), Hu ubiquitin Probeoligonucleotide (1 μM, 5′CGCCGAGATCACCTTTACATTTTCTATCGT-NH2, SEQ IDNO:497), Hu IL-8 Probe (1 μM 5′CCGTCACGCCTCCTCTCAGTTCT-NH2, SEQ IDNO:502), Hu IL-8 Probe Stacker (1 μM, all 2′ O-methyl bases,5′-TTGATAAATTTGGGGTGGAAAGGTTTGGA, SEQ ID NO:503) and the Hu IL-8 INVADERoligonucleotide (0.5 μM, 5′GTGTGGTCCACTCTCAATCAA, SEQ ID NO:504). Thecleaved 5′ flaps from the Primary reaction are Hu ubiquitin 5′ flap(5′CCGCCGAGATCACC, SEQ ID NO:500) and Hu IL-8 5′ flap (5′-CCGTCACGCCTCT,SEQ ID NO:507). At the end of the Primary Reaction, the secondaryreaction mix is added comprising the Hu ubiquitin arrestoroligonucleotide (2.67 μM, all 2′ O-methyl bases,5′ACGATAGAAAATGTAAAGGTGATC, SEQ ID NO:498), Hu ubiquitin Secondary Probe(0.67 μM, RED Dye-CTC-Z28-TTCTCAGTGCG, SEQ ID NO:499), Hu ubiquitinSecondary Target (0.1 μM, the last 3 nt on the 3′ end are 2′ O-methyl;5′ CGCAGTGAGAATGAGGTGATCTCGGCGGU, SEQ ID NO:501), Hu IL-8 arrestoroligonucleotide (2.67 μM, all 2′ O-methyl bases, 5′-AGAACTGAGAGGAGGC,SEQ ID NO:505), Hu IL-8 Secondary probe (0.67 μM, F1-CAC-Z28-TGCTTCGTGG,SEQ ID NO:506), and Hu IL-8 Secondary Target (0.1 μM, the last 3 nt onthe 3′ end are 2′ O-methyl bases, 5′-CCAGGAAGCAAGTGGAGGCGTGACGGU, SEQ IDNO:508).

Example 68 Detection of the Human Cytomegalovirus pol Gene

Example 48 demonstrated the use of the invasive cleavage assay to detecthuman cytomegalovirus sequences in a background of human genomic DNA. Inthis example, another embodiment of the multiple invasive cleavagereaction will be demonstrated for the detection of human cytomegalovirussequence. Example 48 utilized an INVADER and a labeled, primary probeoligonucleotide targeting the region 3104-3061 of hCMV. In this examplebases 2302-2248 of the hCMV genome were targeted. As in previousexamples, a FRET cassette was used to generate signal in the presence ofthe target DNA.

INVADER and probe oligonucleotides were designed as described above todetect the polymerase gene of HCMV (SEQ ID NO:407 and 408,respectively); the FRET cassette is SEQ ID NO:409. Oligonucleotides arediagrammed in FIG. 144. The HCMV target sequence detected by this probeset is SEQ ID NO:410.

Genomic viral DNA was purchased from Advanced Biotechnologies, Inc.(Columbia Md.). The DNA was estimated (but not certified) by personnelat Advanced Biotechnologies to be at a concentration of 170 amol (1×10⁸copies) per microliter. The viral stock was diluted in 10 ng/μl humangenomic DNA to final concentrations of 45.7, 137.2, 411.5 and 1234.6viral copies per microliter. Reactions were performed in MJ 96 wellMULTIPLATE (MJ Research MLP-9601) plates, in triplicate in a finalvolume of μl. Each reaction comprised 12 mM MOPS (pH 7.5), 12 mM MgCl₂,0.5 pmol INVADER oligonucleotide, 10 pmol unlabeled, primary probe, 5pmol One-Piece FRET cassette, 0, 457, 1372, 4115 or 12346 copies of CMVViral DNA, 100 ng human genomic DNA and 60 ng AveFEN1, and water to afinal volume of 2 μl. The MOPS, INVADER oligonucleotide, water, CMVviral DNA and human genomic DNA were combined, overlaid with CHILLOUTliquid wax, and denatured for 5 minutes at 95° C. Reactions were thencooled to 20° C. and MgCl₂, unlabeled primary probe, FRET cassette, andAveFEN1 enzyme were added below the Chill-out layer. The reaction weremixed by pipetting up and down 5-10 times, with care being taken toremain clear of the CHILLOUT layer. Reactions were then incubated for 4hours at 65° C., and plates were read directly on a Cytoflour platereader using the following settings: Excitation=485/20, Emission=530/25,Gain=40 at 10 reads/well.

Results are shown graphically in FIG. 145, with number of copies of thetarget DNA indicated on the horizontal axis and the fluorescence unitsindicated on the vertical axis. These results indicate sensitivedetection of HCMV using the sequential invasive cleavage FRET format.

Selection of oligonucleotides for target nucleic acids other than theanalytes shown here, (e.g., oligonucleotide composition and length), andthe optimization of cleavage reaction conditions in accord with themodels provided here follow routine methods and common practice wellknown to those skilled in the methods of molecular biology.

Example 69 Fen Endonuclease-substrate Complexes

Structure-specific 5′ nucleases play an important role in DNAreplication and repair uniquely recognizing an overlap flap DNAsubstrate and processing it into a DNA nick. The 5′ nucleases uniquelyrecognize an overlap flap DNA substrate. However, in the absence of ahigh resolution structure of the enzyme/DNA complex, the mechanismunderlying this recognition and substrate specificity, which is key tothe enzyme's function, remains unclear. Here we describe athree-dimensional model of the structure-specific 5′ flap endonucleasefrom Pyrococcus furiosus in its complex with DNA. The model is based onthe known X-ray structure of the enzyme and a variety of biochemical andmolecular dynamics data utilized in the form of distance restraintsbetween the enzyme and the DNA. Contacts between the 5′ flapendonuclease and the sugar-phosphate backbone of the overlap flapsubstrate were identified using enzyme activity assays on substrateswith methylphosphonate or 2′-O-methyl substitutions. The enzymefootprint extends 2-4 base pairs upstream and 8-9 base pairs downstreamof the cleavage site, thus covering 10-13 base pairs of duplex DNA. Thefootprint data are consistent with a model in which the substrate isbound in the DNA-binding groove such that the downstream duplexinteracts with the helix-hairpin-helix motif of the enzyme. Moleculardynamics simulations to identify the substrate orientation in this modelare consistent with the results of the enzyme activity assays on themethylphosphonate and 2′-O-methyl-modified substrates. To further refinethe model, 5′ flap endonuclease variants with alanine pointsubstitutions at amino acids expected to contact phosphates in thesubstrate and one deletion mutant were tested in enzyme activity assayson the methylphosphonate-modified substrates. Changes in the enzymefootprint observed for two point mutants, R64A and R94A, and for thedeletion mutant in the enzyme's bA/bB region, were interpreted as beingthe result of specific interactions in the enzyme/DNA complex and wereused as distance restraints in molecular dynamics simulations. While theneither the enzymes of the present invention nor the methods of thepresent invention are limited to any particular mechanism, the modeledstructure indicates that the substrate's 5′ flap interacts with theenzyme's helical arch and that the helix-hairpin-helix motif interactswith the template strand in the downstream duplex 8 base pairs from thecleavage site. This model indicates that there are specific interactionsbetween the 3′ end of the upstream oligonucleotide and the enzyme.

For convenience in comparing the PfuFEN1 enzyme to related enzymes(e.g., FEN enzymes sharing conserved amino acids) amino acids of havingfunction equivalent to any specific amino acids in the PfuFEN1 enzymeare referred to by reference to the Pfu amino acid having that function(e.g., a Y33 “equivalent residue” is an amino acid in a nuclease havingproperties equivalent to those of the Y33 amino acid residue of PfuFEN1.A “Y33 equivalent nuclease” is a cleavage agent such as an endonucleasehaving cleavage activity equivalent to a FEN nuclease comprising a Y33equivalent residue).

Substrates for Enzyme Activity Assays.

FIGS. 149A and 149B shows the overlap flap substrates designed forPfuFEN1 activity assays. The substrate consists of upstream anddownstream oligonucleotides annealed to a template oligonucleotide. The3′ end nucleotide of the upstream oligonucleotide, a thymine in thissubstrate, overlaps with the first base pair, G-C, of the downstreamduplex thus creating the optimal substrate for the structure-specific 5′nucleases (Kaiser et al., 1999). Although the overlapping nucleotide isimportant for enzyme activity, it does not need to be complementary tothe template and can be mismatched (Lyamichev et al., 1999). Thedownstream oligonucleotide consists of two regions: a 5′ flap regionthat is non-complementary to the template, and a complementarytemplate-specific region. The substrate is cleaved by thestructure-specific 5′ nucleases after the first base pair of thedownstream oligonucleotide, releasing the 5′ flap with one nucleotide ofthe template-specific region.

The following nomenclature was used to describe the positions of sugarand phosphate residues in the substrate. Each nucleoside is denoted bythe corresponding base; a t, u or d subscript is used to indicatetemplate, upstream, or downstream oligonucleotide, respectively; and anumber is used to designate the position of this nucleoside relative tothe 5′ end of the corresponding oligonucleotide. The phosphates arereferred to as P and carry the same subscript and number as the adjacent3′ nucleoside. For example, the first base-paired nucleoside C at the 5′end of the downstream oligonucleotide is referred to as Cd5 and thecleavable phosphodiester linkage as P_(d)6 (see FIG. 149A forreference).

Enzyme Activity Assay on Modified Substrates.

Methylphosphonate walking experiments. Methylphosphonate substitutionswere used to probe ionic interactions between the PfuFEN1 enzyme andphosphates in the overlap flap substrate. The phosphodiester andmethylphosphonate linkages are diagrammed in FIG. 150. The non-modified(natural) substrate and substrates modified with a singlemethylphosphonate at each position of the template, upstream, ordownstream oligonucleotide were cleaved with PfuFEN1, and the cleavageproducts were analyzed by gel electrophoresis to determine the initialcleavage rates as described in “Materials and Methods” (FIG. 151). The50 nM substrate concentration used in these experiments was at least4-fold lower than the PfuFEN1 KM value for the natural substrate whichis expected to have the lowest KM among all the substrates and,therefore, the initial cleavage rates were used to calculate the kcat/KMvalues. The kcat/KM values normalized for the natural substrate areshown in FIG. 152. Because of ˜25% variability of kcat/KM, onlymethylphosphonate substitutions that decreased the relative kcat/KMvalues by 25% or more were considered to affect the enzyme/substrateinteractions. Using these criteria, we hypothesized that positionsP_(d)5-P_(d)8 and Pu19 in the downstream and upstream oligonucleotides,respectively, were involved in ionic interactions with PfuFEN1. In thetemplate strand, positions P_(t)12-P_(t)13, P_(t)17-P_(t)19 andP_(t)21-P_(t)22 were identified as three areas of interaction withPfuFEN1. Similar methylphosphonate modification results were obtainedwith the overlap flap substrate for AfuFEN1 and MjaFEN1 (data notshown).

2′-O-Methyl Walking Experiments.

The substitution of 2′-O-methyl at the 2′ position of deoxyribose shouldintroduce steric clashes at sites of close contact with PfuFEN1. Similarto the methylphosphonate walking experiments, we used PfuFEN1 to cleavesubstrates in which the template, upstream or downstream oligonucleotidecontained a single 2′-O-methyl substitution at each deoxyribose todetermine the initial cleavage rates and calculate kcat/KM values (FIG.153). Significant decreases in kcat/KM were demonstrated for 2′-O-methylsubstitutions at positions Td4 and Cd5 in the downstreamoligonucleotide, at the first and second nucleosides from the 3′ end ofthe upstream oligonucleotide, Tu19 and Cu18, and at two regions,Ct11-Ct12 and Gt19-Gt20, in the template oligonucleotide. Overall, the2′-O-methyl substitutions identified the same regions of interactionwith PfuFEN1 as did the methylphosphonate walking experiments (compareFIGS. 152 and 153). Particularly, both 2′-O-methyl substitutions atCt11-Ct12 and methylphosphonate substitutions at P_(t)12-P_(t)13inhibited cleavage by PfuFEN1, thus supporting the conclusion that thetemplate strand in downstream duplex is recognized by PfuFEN1 8-9 basepairs from the overlap.

Orientation of the DNA Substrate in PfuFEN1/DNA Complex.

The first goal of MD simulations was to test two possible orientationsof the substrate in the DNA-binding groove of PfuFEN1 and to select theorientation consistent with the methylphosphonate and 2′-O-methylwalking experiments. In orientation I, the downstream duplex was placedin the DNA-binding groove close to the HhH motif of PfuFEN1 (residues223-256) as originally suggested by Kaiser et al. (Kaiser et al., 1999).In orientation II, the substrate was rotated 180° and placed in theopposite direction relative to the downstream and upstream duplexes,similar to the model proposed for 5′-3′ exonuclease from bacteriophageT5 (Ceska et al., 1996). For both orientations, the only restraintimposed on the enzyme-DNA interactions was a distance of 3.5±1.5 Åbetween magnesium ion M-1 and the cleavable phosphodiester bond P_(d)6(minimal restraints model).

MD simulations predict different structures for orientations I and II(FIG. 154). In particular, in orientation I, the template strand (green)in the downstream duplex interacts with the HhH motif (red) 6 to 9 basepairs from the overlap in the Ct11-Ct14 region. On the other hand, inorientation II, PfuFEN1 enzyme contacts with the template strand in thedownstream duplex are confined to the Gt16-Gt19 region. The interactionspredicted in orientation I, but not those in orientation II, areconsistent with the results of enzyme activity assays on themethylphosphonate and 2′-O-methyl-modified substrates, which suggestthat the P_(t)12-P_(t)13 phosphates and Ct11-Ct12 deoxyribose residuesare involved in contacts with PfuFEN1.

Mutational Analysis of Interactions in the PfuFEN1/DNA Complex.

To provide additional support for orientation I and to identify specificPfuFEN1/DNA interactions, amino acids from four different regions in theDNA-binding groove were selected for mutational analysis. Group 1includes the Y33, Q34, R40, and R64 amino acids located in the a2 and a3helixes of PfuFEN1 (Hosfield et al., 1998b) that presumably interactwith the upstream duplex in the orientation I model. Group 2 includesthe R94, R95, and R98 amino acids from the C-terminus of helical archregion, which are predicted to interact with P_(d)5 of the probe strand.Group 3 includes all positively charged amino acids K193, R194, K195,K199, and K206 in the PfuFEN1 bA/bB hairpin (Hosfield et al., 1998b) andthe Q172 and R186 amino acids, which are predicted to interact with theGu15-Cu18 region of the upstream strand. Also, this group included thePfuM3 mutant in which the bA/bB hairpin was replaced with a shorter loopof amino acids Lys-Glu-Met from MjaFEN1 (residues 192-194). This loopwas identified as a counterpart of the bA/bB hairpin in a structuralalignment of PfuFEN1 and MjaFEN1. Group 4 includes the K243, K248, andK249 amino acids of the HhH motif and amino acids K266 and Q267 of thea12/a13 hairpin (261-280) predicted to interact with P_(t)11-P_(t)13 ofthe template strand.

Individual amino acids from all four groups were substituted withalanine to produce PfuFEN1 point mutants. We assumed that amethylphosphonate substitution at a phosphate forming an ionic contactwith a positively charged amino acid would have a smaller effect on therelative activity of the PfuFEN1 variant if this amino acid was changedto an uncharged amino acid, e.g. alanine. Therefore, the PfuFEN1 pointmutants were tested in the activity assay on methylphosphonate-modifiedsubstrates, and relative kcat/KM values obtained for each mutant werecompared to those obtained using the wild type PfuFEN1 on themethylphosphonate-modified substrates.

Among Group 1 mutants, the Y33A mutation produced an inactive enzyme,and both the Q34A and R40A mutations showed no significant effect onrelative kcat/KM for all methylphosphonate-modified substrates (data notshown). However, the R64A mutant produced a kcat/KM profile on thetemplate strand that was different from that of wild type PfuFEN1(compare FIGS. 155A and 152A). A significant increase in relativekcat/KM was observed for the R64A mutant on the P_(t)21methylphosphonate substrate. Interestingly, the R64A mutation decreasedrelative kcat/KM values for the P_(t)11, P_(t)23 and P_(t)24methylphosphonate substrates.

In Group 2, the R95A mutant showed no activity, and the R98A mutantshowed no effect on the kcat/KM profiles on all methylphosphonate DNAsubstrates (data not shown). The R94A mutation had no effect on thekcat/KM profiles on the template and upstream strands. However, in thedownstream strand, a significant increase in relative kcat/KM wasobserved on the P_(d)5 and P_(d)6 methylphosphonate substrates (compareFIGS. 155B and 152C). The increase in kcat/KM of the R94A mutant on theP_(d)6 methylphosphonate substrate is surprising since P_(d)6 is thephosphodiester linkage cleaved by PfuFEN1, and its modification with amethylphosphonate is expected to have a strong inhibitory effect onenzyme activity. However, prolonged treatment of the Pd6methylphosphonate substrate with the R94A mutant showed onlyapproximately 50% substrate cleavage suggesting that only one of twostereoisomers was cleaved by the mutant.

None of the Q172A, R186A, K193A, R194A, K195A, K199A, or K206A pointmutants included in Group 3 showed a significant effect on the kcat/KMprofile of any of the methylphosphonate substrates (data not shown). ThePfuM3 mutant demonstrated an increase in relative kcat/KM on theP_(t)17, P_(t)18, P_(t)21, and P_(t)22 methylphosphonate substratescompared to the wild type PfuFEN1 (FIG. 155C). Finally, no differencesin the kcat/KM profiles on any of the methylphosphonate substrates wereobserved for the Group 4 point mutants K243A, K248A, K249A, K266A, Q267Aand the K248A/K249A double mutant.

To confirm the kcat/KM results, individual KM and kcat values for theR64A, R94A, PfuM3 mutants, and wild type PfuFEN1 on the P_(t)12,P_(t)21, P_(d)5, and Pu19 methylphosphonate substrates and the naturalsubstrate were determined as described in “Materials and Methods” (Table1). Comparison of the wild type PfuFEN1 and the R64A mutant shows thatthe P_(t)21 methylphosphonate substitution increases the KM value ofPfuFEN1 approximately 18-fold but has virtually no effect on the KM ofthe R64A mutant. The P_(d)5 methylphosphonate substitution does notpractically affect the kinetic parameters for the R94A mutant; however,it increases the wild type PfuFEN1 KM value approximately 8-fold anddecreases the kcat value 9-fold. Finally, the P_(t)21 modificationincreases the KM value for PfuM3 3-fold compared to an approximately18-fold increase for PfuFEN1. A similar decrease in kcat is observed forboth enzymes. Overall, the individual KM and kcat values shown below inTable 6 agree with the kcat/KM results obtained in the enzyme activityassays.

This mutational analysis supports orientation I of the PfuFEN1/DNAcomplex and indicates that R94 and R64 contact P_(d)5 and P_(t)21,respectively, and that the bA/bB loop contacts the P_(t)17-P_(t)18 andP_(t)21-P_(t)22 regions in the template strand.

Table 6. Kinetic parameters¹ of the wild type and mutant PfuFEN1proteins on the methylphosphonate-modified substrates.

DNA Substrate² Enzyme Natural DNA P_(t)12 P_(t)21 P_(d)5 P_(u)19 PfuFEN1K_(M) (μM) 0.20 ± 0.07 1.02 ± 0.49 3.59 ± 1.09  1.61 ± 0.18 1.49 ± 0.22k_(cat) (min⁻¹) 62.1 ± 22.3  1.8 ± 0.8  7.6 ± 2.1  7.1 ± 0.8 18.3 ± 1.6R64A K_(M) (μM) 0.43 ± 0.06 1.04 ± 0.90 0.38 ± 0.05  1.89 ± 0.73 1.48 ±0.50 k_(cat) (min⁻¹) 21.8 ± 2.3  2.7 ± 2.3 × 10⁻³  7.4 ± 0.9  0.5 ± 0.210.5 ± 3.0 × 10⁻³ R94A K_(M) (μM) 0.21 ± 0.03 0.69 ± 0.38 2.80 ± 0.32 0.25 ± 0.09 1.46 ± 0.58 k_(cat) (min⁻¹) 12.0 ± 1.0 × 10⁻³  1.1 ± 0.6 ×10⁻³  3.4 ± 0.4 × 10⁻³  24.0 ± 7.0 × 10⁻³  .5 ± 3.7 × 10⁻³ PfuM3 K_(M)(μM) 0.90 ± 0.35 7.65 ± 2.60 2.89 ± 1.44 12.95 ± 7.29 8.21 ± 1.27k_(cat) (min⁻¹)  5.1 ± 1.9  0.1 ± 0.1  1.0 ± 0.5  5.5 ± 3.0 × 10⁻³  2.1± 0.3 ¹K_(M) and k_(cat) parameters were determined in 50 mM KCl, 7.5 mMMgCl₂, 10 mM MOPS at 51° C. ²Position of the methylphosphonate modifiedphosphodiester linkages.MD Simulations of the PfuFEN1/DNA Complex with Residue-specificRestraints.

The interactions revealed by the mutational analysis were used toimprove the structural model of the PfuFEN1/DNA complex by introducingdistance restraints between specific residues of the enzyme and the DNAinto MD simulations. In the minimal restraints model (FIG. 154A), thedistance between the center of mass the amino group of R64 and thephosphorous in P_(t)21 is 17.6 Å and those for the R94-P_(d)5 andR94-P_(d)6 interactions are 11.7 Å and 14.9 Å, respectively. Based onthe proposed R64A-P_(t)21 and R94A-P_(d)5 contacts, we introduced adistance restraint of <8 Å between the center of mass of the aminogroups of the side chain of R64 and R94 and the phosphorous atom ofP_(t)21 and P_(d)5, respectively.

Although the mutational analysis did not predict interactions betweenthe positively charged amino acids of the PfuFEN1 HhH motif and the DNA,it was assumed that the HhH motif contacts the P_(t)13 and P_(t)12phosphates in the template strand based on the methylphosphonate walkingexperiments (FIG. 152A). To define restraints for the HhH motif, we usedco-crystal structures for human polymerase b (PDB accession 1BPY)(Sawaya et al., 1997) and E. coli 3-methyladenine DNA glycosylase II(IDIZ) (Hollis et al., 2000) that demonstrate interactions of thecognate HhH motifs with double-stranded DNA substrates. The HhHinteractions in these structures occur through hydrogen bonds of <3.3 Åbetween oxygen atoms of two adjacent DNA phosphates and the backbonenitrogens of the G105, G107, 109, and A110 residues in polymerase b andthe G214, G216, and T219 residues in glycosylase. The correspondingresidues in the PfuFEN1 HhH motif, G244, G246, K248, and K249, wereidentified by structure-based sequence alignment (Shao & Grishin, 2000).In the minimal restraints model, the distances between the backbonenitrogens of G244, G246, K248, and K249 and the nearest oxygen ofP_(t)13 or P_(t)12 are 10.9, 15.6, 18.2, and 18.2 Å, respectively. Tointroduce hydrogen bond interactions observed in the co-crystals, thedistances between the G244, G246, K248, and K249 backbone nitrogens anda center of mass of phosphorus and two nonbridging oxygens of P_(t)13and P_(t)12 were restrained to 3.5±1.0 Å.

FIG. 156 shows a 5-ns MD structure of the PfuFEN1/DNA complex usingresidue-specific restraints. In the residue-specific model, thesubstrate moved deeper in the active-site DNA-binding groove comparedwith the minimal restraints model. For instance, the distance betweenthe center of mass the arginine amino groups and phosphorous inR64-P_(t)21, R94-P_(d)5, and R94-P_(d)6 pairs is decreased to 3.8, 3.8,and 4.3 Å, respectively. The DNA also moved closer to the HhH motif toform hydrogen bonds O2-P_(t)13-G244, O2-P_(t)12-G246, O1-P_(t)12-K248,and O1-P_(t)12-K249 of 2.77, 3.7, 3.3, and 3.0 Å, respectively. Inaddition, positively charged residues of the HhH motif demonstrateinteractions with P_(d)8 in the downstream strand and with the majorgroove of the downstream duplex in the Ct 11-Ct 13 region. The upstreamand downstream duplexes are in the B-form conformation and form an angleof approximately 40° between duplex axes at the overlap. The M-1magnesium ion interacts with the cleavable phosphodiester bond P_(d)6and the M-2 ion which is not bound by any restraints interacts with theP_(d)6 and P_(d)7 phosphates. Interactions predicted by the finalstructure are summarized in FIG. 157.

In this work, we have modeled a three-dimensional structure of thePfuFEN1 structure-specific 5′ nuclease in its complex with DNA using MDsimulations guided by enzyme-DNA distance restraints derived fromexperimental data. Regions of the overlap flap substrate involved inspecific contacts with PfuFEN1 were identified using methylphosphonateand 2′-O-methyl walking experiments. Most contacts were observed closeto the cleavage site, however, an additional region of bothelectrostatic and steric interactions was identified on the templateoligonucleotide at positions P_(t)12, P_(t)13, Ct11, and Ct12 (FIG.149A). The asymmetry of the footprint, which extends 9 base pairs intothe downstream duplex and only 2 base pairs into the upstream duplex,agrees well with analyses of enzyme activity on overlap flap substratesof different length (Kaiser et al., 1999) and phosphate ethylationinterference experiments with a flap substrate and the 5′ nucleasedomain of DNA polymerase I from E.coli (Xu et al., 2001).

The PfuFEN1 footprint was used to determine the orientation of thesubstrate in the DNA-binding groove. Originally proposed models for the5′ nuclease/DNA complex suggested that the upstream duplex of the flapsubstrate interacts with the HhH motif of the 5′ nuclease enzymes (Ceska& Sayers, 1998; Ceska et al., 1996; Hwang et al., 1998). However, basedon the minimal substrate length requirements, Kaiser et al. (1999)proposed that the HhH motif interacts with the downstream duplex of theoverlap flap substrate. A similar model was also proposed by Dervan etal. (2002) from kinetic and binding characteristics of T5 5′ nucleasemutants using hairpin-like substrates containing only a downstreamduplex. MD simulations using only minimal restraints specifying distancebetween the cleavable linkage P_(d)6 and M-1 magnesium ion showed thatonly orientation I, in which the downstream duplex interacts with theHhH motif, was consistent with the asymmetric pattern of the PfuFEN1footprint and was proposed as a minimal restraints model of thePfuFEN1/DNA complex (FIG. 154A).

Binding of DNA substrates often induces significant conformationalchanges in protein structure. For example, comparison of the apo and DNAcomplex structures of the Klenow fragment of E.coli DNA polymerase Idemonstrates that the thumb region of the protein moves as much as 12 Åto accommodate the DNA (Beese et al., 1993). There is no indication ofinduced conformational changes in the minimal restraints model, and itis unlikely that such changes, which may occur at a microsecond or evenmillisecond time range (Katahira et al., 2001), would be observed innanosecond-long MD models. The importance of these considerations can beillustrated by the nature of the interactions between the HhH motif andthe downstream duplex predicted by the minimal restraints model to occurthrough ionic contacts between the side chains of K248 and K249 and theP_(t)12-P_(t)13 phosphates. These interactions contradict the co-crystalstructures of human polymerase b (Sawaya et al., 1997) and E. coli3-methyladenine DNA glycosylase II (Hollis et al., 2000), demonstratingthat HhH motif interacts with double-stranded DNA by forming hydrogenbonds between the backbone nitrogens of conserved HhH residues andoxygens of two adjacent DNA phosphates. The contacts suggested from theco-crystal structures would require that the downstream duplex in theminimal restraints model move closer to the PfuFEN1 HhH motif and formspecific hydrogen bonds with backbone nitrogens of G244, G246, K248, andK249.

To experimentally determine specific interactions in the PfuFEN1/DNAcomplex, we used enzyme activity assays with mutant PfuFEN1 enzymes onmethylphosphonate-containing substrates. The basis for this approach hasbeen previously proven for specific steric interactions between the MS2bacteriophage coat protein and its cognate RNA hairpin (Dertinger etal., 2001). The methylphosphonate profiling of the PfuFEN1 mutantsidentified specific ionic contacts between R94 and P_(d)5 and betweenR64 and P_(t)21. A similar approach was previously used to identifyspecific contacts between the 5′ nuclease domain of DNA polymerase Ifrom E. coli and a flap substrate (Xu et al., 2001) withmethylphosphonate substitutions studied in the downstream strand atpositions corresponding to P_(d)3, P_(d)4, P_(d)5 and P_(d)7. Theauthors observed a similar inhibitory effect on the cleavage rate ofsubstrate modified at positions analogous to P_(d)5 and P_(d)7 and,based on mutational analysis, suggested that these phosphates interactwith Y77, K78, and R81 and R20 of the E. coli 5′ nuclease domain,respectively. Alignment of the E. coli 5′ nuclease domains and TaqExosequences followed by three-dimensional structure alignment of TaqExoand PfuFEN1 suggests that Y77, K78, R81, and R20 of the E. coli 5′nuclease domain correspond to R94, R95, R98, and Q34 of PfuFEN1,respectively. In this work we show that R94 contacts P_(d)5, but theR95A mutation results in an inactive enzyme and both the Q34A and R98Amutations have no significant effect on the methylphosphonate profiling.These differences can be explained by a misalignment of the E. coli 5′nuclease domain and PfuFEN1 due to low sequence similarity anddifferences in the binding characteristics of the overlap flap and flapsubstrates. Introduction of the residue-specific restraints moved thesubstrate deeper in the DNA-binding groove compared to the minimalrestraints model. Superposition of enzyme structures in the minimal andfinal models shows that the P_(t)13 and P_(t)12 phosphates in thedownstream duplex move by ˜20 Å and the upstream duplex moves by 17 Å onaverage. The tighter contacts between the DNA and enzyme became possiblebecause of conformational changes in the enzyme structure: the Ca atomsof the bA/bB loop (192-206) moved by 11 Å on average with a maximumdistance of 16.5 Å observed for P197. The final PfuFEN1/DNA model waschallenged to explain differences between the methylphosphonate and2′-O-methyl PfuFEN1 footprints. The latter identifies stericinteractions that have not been used to specify the distance restraintsin MD simulations. In the downstream strand, these differences aremanifested by an effect of the P_(d)6, P_(d)7, and P_(d)8methylphosphonate substitutions on kcat/KM and the lack of such aneffect for the Ad6 and Ad7 2′-O-methyl substitutions (FIG. 149A). Themodeled structure explains. this pattern by the existence of contactsbetween P_(d)6-P_(d)8 and the magnesium ions and K248 whereas the Ad6and Ad7 nucleosides are exposed in solution outside the DNA-bindinggroove and do not contact PfuFEN1. Conversely, the Gt20 and Gt192′-O-methyl modifications, but not the P_(t)20 and P_(t)19methylphosphonate modifications, affect kcat/KM. Again, the modelpredicts that the Gt20 and Gt19 nucleosides face the DNA-binding groove,and the 2′-O-methyl substitutions at these positions are likely to causea steric clash with PfuFEN1. To the contrary, no specific interactionsare predicted for P_(t)20 and P_(t)19.

There are several interactions suggested from the PfuFEN1 footprintsthat cannot be explained by the final model. For example,methylphosphonate substitutions at P_(t)17 and P_(t)18 decrease kcat/KM(FIG. 152A); however, no contacts involving these phosphates arepredicted in the model. Potential candidates for these contacts includea group of positively charged lysines 87, 89, 93 and arginines 94, 95,98, 106 in the helical arch region; however, the methylphosphonateprofiling does not support this hypothesis for three tested amino acids,R94, R95, and R98. Interaction between the helical arch and the P_(t)17and P_(t)18 phosphates might be only possible as a result of largesubstrate-induced conformational changes in the helical arch, such aspreviously suggested for human FEN1 from Fourier transform infraredspectroscopy demonstrating an increase in helicity of the helical archregion upon DNA binding (Kim et al., 2001). Some interactions suggestedin the model and supported by PfuFEN1 footprint data still need to beconfirmed by mutational analysis. For example, according to the model,Pu19 forms ion pairs of 2.9 Å, 3.0 Å, and 3.9 Å with K104, K111, andR40, respectively, and the methylphosphonate substitution at Pu19decreases kcat/KM value of PfuFEN1 (FIG. 152B). Only one of these aminoacids, R40, was tested in the methylphosphonate profiling experiments,but showed no interaction with Pu19.

The 3′ terminal nucleotide of the upstream strand is an importantelement in substrate recognition by FEN1 enzymes. The final modelpredicts that distance between centers of the 3′ nucleotide and the Y33amino acid aromatic rings measured over 100 snapshots taken from thelast 1 ns of 5 ns MD simulation is ˜6±2 Å. This distance corresponds toa maximum of distance distribution for aromatic amino acid pairs inX-ray crystal structures of nonhomologous proteins (McGaughey et al.,1998) suggesting stacking interactions between the 3′ nucleotide and theY33 residue. In support of this conclusion, Y33 is highly conserved inthe FEN1 family, and substitution of this amino acid with alanine orglutamic acid inactivates PfuFEN1, but its substitution with anotheraromatic amino acid, phenylalanine, reduces PfuFEN1 activity only by 50%(data not shown). The exact position of the 5′ flap, which according tothe threading model should be within the helical arch of PfuFEN1(Lyamichev et al., 1993; Murante et al., 1995), is not defined in thefinal model. During the dynamics the flap moves in and out of thehelical arch due to the high flexibility of both the helical arch andthe 5′ flap, thus making it difficult to interpret its specificcontacts. It is possible that conformational changes suggested for thehelical arch should be included in the model to determine specificinteractions of the 5′ flap and the 3′ terminal nucleotide of theupstream strand.

Significant progress in understanding of the mechanism of lagging strandsynthesis in eukaryotes led to a model describing the synthesis as aseries of highly coordinated steps performed by the Okazaki fragmentprocessing complex which includes FEN1 [Maga, 2001; Bae, 2001]. It isthought that the synchronized product release in one step and itsbinding as a substrate in the next step is governed by substratespecificity of the proteins constituting the complex. FEN1 plays acritical role in the transition from the strand displacement stepperformed by eukaryotic polymerase d to the 5′ flap removal step and inthe passing the nick substrate to DNA ligase I. The first detailed modelof PfuFEN1/DNA complex proposed in this work is an important step inunderstanding the organization of Okazaki fragment processing complexand dynamics of complex rearrangement during the lagging strandmaturation. In this work we employed MD simulations in conjunction withexperimentally determined restraints to develop the most detailed modelof the PfuFEN1/DNA complex to date. The model provides the correctorientation of the overlap substrate in active-site DNA-binding grooveof PfuFEN1 and describes specific interactions supported by the majorityof the methylphosphonate and 2′-O-methyl walking results and mutationalanalyses of PfuFEN1. The modeled PfuFEN1/DNA structure may be furtherimproved by incorporating new experimental data. Interactions predictedby the model can also be used to understand the mechanism of Okazakifragment processing and DNA repair and to develop new variants of FEN1enzymes for biotechnological applications.

Materials and Methods

DNA synthesis and purification: DNA oligonucleotides were synthesizedwith an Expedite 8909 synthesizer (PerSeptive Biosystems) using standardphosphoramidite chemistry. All phosphoramidites includingtetracholorofluorecsein (TET) dye, 2′-O-methyl, and methylphosphonatephosphoramidites were purchased from Glen Research, Sterling, Va.Synthesis and deprotection were performed according to the vendor'srecommended procedures and further purification was performed byelectrophoresis on a 20% denaturing acrylamide gel as described (Kaiseret al., 1999). Methylphosphonate oligonucleotides were used withoutseparation of Rp and Rs isomers. The purity of all oligonucleotides usedin enzyme activity assays was analyzed by capillary electrophoresis on aP/ACE MDQ CE system (Beckman) with mPAGE-5 capillaries (AgilentTechnologies, Palo Alto, Calif.). Oligonucleotide concentrations weredetermined from the absorption at 260 nm using extinction coefficientsfor nucleosides and dinucleotide monophosphates (Gray et al., 1995).

Mutagenesis. The Y33A, Q34A, R40A, R64A, R94A, R95A, R98A, Q172A, R186A,K193A, R194A, K195A, K199A, K206A, K243A, K248A, K249A, K266A, and Q267Amutants were prepared by site-directed mutagenesis with a Quikchange kit(Stratagene) using 30 cycles of PCR amplification on the pTrc99a-PfuFEN1plasmid DNA (Kaiser et al., 1999) with 200 nM mutagenic primers. AfterDpn I digestion the amplified DNA was transformed into E. coli DH5á byelectroporation. The presence of desired mutations was verified bysequencing. The PfuM3 deletion variant was prepared by replacing aminoacids GKRKLPGKNVYVEIK of (SEQ ID NO:520) of PfuFEN1 at position 192-206with amino acids KEM of MjaFEN1 (192-194) using recombinant PCR. The 5′half of the PfuM3 gene was amplified with 5′-TGTGGAATTGTGAGCGG (SEQ IDNO:521)(pTrcFwd) and 5′-CTCTGGCATCTCCTTTGTTATTGTTAAGTTTCTAAC (SEQ IDNO:522) primers and the 3′ half was amplified with5′-ACAAAGGAGATGCCAGAGTTGATAATTTTGGAGGAAG (SEQ ID NO:523) and5′-TAATCTGTATCAGGCTG (SEQ ID NO:524)(pTrcRev) primers. The DNA productswere purified from a 1% agarose gel using a Qiaquick kit (Qiagen) andused in PCR reaction with the pTrcFwd and pTrcRev primers. The amplifiedDNA was gel purified and cloned into pTrc99a vector as described (Kaiseret al., 1999).

Enzyme expression and purification. The plasmids carrying PfuFEN1variants were transformed into E. coli BL21 (Novagen) for expression andpurification of the proteins was performed as described (Kaiser et al.,1999). At the final step of purification the proteins were dialyzed anddiluted in a buffer containing 50% glycerol, 20 mM Tris-HCl, pH 8, 50 mMKCl, 0.5% Tween 20, 0.5% Nonidet P40, 100 mg/mL BSA.

Kinetic parameter measurement and enzyme activity assays. Kineticparameters, KM and kcat, for the wild type and mutant PfuFEN1 enzymeswere determined with the substrate shown in FIG. 149A. Reactions wereprepared by mixing the downstream, upstream and templateoligonucleotides at 5 mM concentration each in a buffer containing 100mM MOPS, 75 mM MgCl2 and heating the sample to 95 oC for 1 minute andslow cooling to room temperature. The annealed substrate was stored at−20 oC. The kinetics experiments were performed at different substrateconcentrations from 10 to 800 nM and constant enzyme concentrationbetween 0.1 and 100 nM, depending on the specific activity of aparticular enzyme. Enzyme/substrate mixtures were prepared in 100 mL 10mM MOPS, pH 7.5, 50 mM KCl, 7.5 mM MgCl2, 10 ng/mL tRNA on ice, and thereactions were started by incubation at 51 oC. 10 mL aliquots of eachreaction were then transferred into pre-chilled tubes containing 15 mL95% formamide, 10 mM EDTA, and 0.02% methyl violet blue (Sigma-Aldrich)at time points of 5, 10, 15, 30, and 60 min. 5 mL aliquots of thestopped reactions were then analyzed by gel electrophoresis to obtaininitial rates of the cleavage reactions as described (Kaiser et al.,1999). The KM and kcat values were calculated from the initial ratesusing the Michaelis-Menten equation. The enzyme activity assay used forfast measurement of kcat/KM values for wild-type and mutant PfuFEN1enzymes was performed in 10 mL 10 mM MOPS, pH 7.5, 50 mM KCl, 7.5 mMMgCl2, 10 ng/mL tRNA including 0.1 to 5 nM enzyme and 50 mM natural ormodified substrate. The reactions were assembled on ice and thenincubated at 51° C. for periods of time sufficient to reach 5 to 20 %substrate cleavage. The kcat/KM values were calculated as the initialcleavage rate divided by the enzyme and initial substrate concentrationsunder the assumption that the initial substrate concentration was muchlower than KM.

Molecular Modeling. PfuFEN1 structure coordinates used in MD simulationswere obtained from the PDB database, accession number 1B43 (Hosfield etal., 1998b). To reduce computation time, the C-terminus residues 290-340of PfuFEN1 were omitted. A B-form DNA duplex of the overlap flapsubstrate (FIG. 149B) was generated using the Biopolymer software(Insight II—Accelrys, Inc.). To assemble the initial complex, thesubstrate was manually placed in the active-site DNA-binding groove ofPfuFEN1 with an average distance of 3 to 15 Å from the enzyme with thecleavable phosphodiester linkage facing the M-1 and M-2 metal ions. MDsimulations were preformed with AMBER 6.0 software (Case et al., 1999;Weiner et al., 1984). A 2 fs time-step was employed and SHAKE was usedfor bonds involving hydrogens. The generalized Born (GB) solvation modelwas used with a non-bonded interactions cutoff distance of either 8 Å or12 Å and 0.2 M monovalent salt concentration (Onufriev et al., 200; Tsui& Case, 2000a; Tsui & Case, 2000b). A 1 ns MD required ˜6 days ofcomputation on a cluster of 4 computers with dual 1.5 GHz Athlon (AMd.)processors and Linux Mandrake 8.1 operating system. The MD protocol usedin this work included energy minimization (EM), simulated annealing(SA), and production stages. During the EM stage, the initialPfuFEN1/DNA complex assembly was subjected to 200 steps of conjugategradient energy minimization to relax energetically unfavorableinteractions. Following the EM stage, an SA stage was performed duringwhich the system was gradually heated from 0 to 500 K for 6 ps using aheat bath time coupling constant of 2 ps, then the system was slowlycooled to 300 K over a period of 14 ps while gradually decreasing theheat bath constant to 0.5 ps. During the SA stage, all enzyme residuesexcept the two magnesium ions, the helical arch (residues 75-127), thebA/bB loop (184-210), and the HhH motif (223-256) were constrained totheir crystallographic positions using a harmonic potential forceconstant kc of 5 kcal/mol/Å2. Additionally, DNA residues exceptAu17-Tu19, Td1-Ad7, and Tt17-Tt21 were restrained using a flat-wellharmonic potential of 2.25±0.50 Å for Watson-Crick hydrogen bonds anda=−65±60°, b=−150±45°, g=40±45°, d=120±90°, e=170±60°, z=−70±90°, andc=−125±90° for dihedral angels. Finally, flat-well harmonic restraintscalled ‘minimal restraints’ were used to define distance of 3.50±1.50 Åbetween the M-1 ion and the cleavable phosphodiester linkage P_(d)6.During the SA stage, the restraint penalties were gradually introducedby increasing their harmonic potential force constant kr from 0 to 15kcal/mol/Å2 over the 6 ps heating steps and then to 20 kcal/mol/Å2 overthe 14 ps cooling steps to allow for slow relaxation and preventabnormal changes in the enzyme or DNA structure. The production stagewith minimal restraints was performed using a temperature couplingconstant of 1.0 ps, kc of 1 kca1/mol/Å2 and kr of 20 kcal/mol/Å2 at 300K for 1 ns. ‘Residue-specific’ restraints included the minimalrestraints and an additional set of distance restraints definingspecific contacts between the substrate and the enzyme (see “Results”).Using the final structure of the 1 ns minimal restraints trajectory asstarting coordinates, MD simulations with residue-specific restraintswere performed with 20 ps SA followed by 1 ns production stage (seeabove) and 4 ns of additional simulation during which the enzymeconstraints were removed for all residues. The final structure wassubjected to 500 steps of conjugate gradient energy minimization. Cutoffdistances of either 8 Å or 12 Å for non-bonded interactions was used fortwo initial 1 ns production dynamics with residue-specific restraints.Both cutoff distances produced similar structures with RMSD for heavyatoms of ˜1.2 Å. To speed up computation, the 8 Å cutoff distance wasused for the 4 ns production dynamics with residue-specific restraints.The average heavy atoms RMSD between consecutive 1 ps structures of 1 nsMD simulations converged to <1.5 Å RMSD after 400-500 ps.

REFERENCES

-   Beese, L. S., Derbyshire, V. & Steitz, T. A. (1993). Structure of    DNA polymerase I Klenow fragment bound to duplex DNA. Science 260,    352-5.-   Bhagwat, M., Meara, D. & Nossal, N. G. (1997). Identification of    residues of T4 RNase H required for catalysis and DNA binding. J    Biol Chem 272, 28531-8.-   Bomarth, C. J., Ranalli, T. A., Henricksen, L. A., Wahl, A. F. &    Bambara, R. A. (1999). Effect of flap modifications on human FEN1    cleavage. Biochemistry 38, 13347-13354.-   Botfield, M. C. & Weiss, M. A. (1994). Bipartite DNA recognition by    the human Oct-2 POU domain: POUs-specific phosphate contacts are    analogous to those of bacteriophage lambda repressor. Biochemistry    33, 2349-2355.-   Cantor, C. R. & Schimmel, P. R. (1980). In Biophysical Chemistry.    Part III: The behavior of biological macromolecules, pp. 1036-1037.    W.H. Freeman and company, New York.-   Case, D. A., Pearlman, D. A., Caldwell, J. C., III, Cheatham, T. E.,    Ross, W. S., Simmerling, C. L., Darden, T. A., Merz, K. M.,    Stanton, R. V., Cheng, A. L., Vincent, J. J., Crowley, M., Tsui, V.,    Radmer, R. J., Duan, Y., Pitera, J., Massova, I., Seibel, G. L.,    Singh, U. C., Weiner, P. K. & Kollman, P. A. (1999). AMBER 6.    University of California, San Francisco.-   Ceska, T. A. & Sayers, J. R. (1998). Structure-specific DNA cleavage    by 5′ nucleases. Trends Biochem Sci 23, 331-6.-   Ceska, T. A., Sayers, J. R., Stier, G. & Suck, D. (1996). A helical    arch allowing single-stranded DNA to thread through T5 5′-    exonuclease. Nature 382, 90-93.-   Dertinger, D., Dale, T. & Uhlenbeck, O. C. (2001). Modifying the    specificity of an RNA backbone contact. J Mol Biol 314, 649-654.-   Dertinger, D. & Uhlenbeck, O. C. (2001). Evaluation of    methylphosphonates as analogs for detecting phosphate contacts in    RNA-protein complexes. Rna 7, 622-631.-   Dervan, J. J., Feng, M., Patel, D., Grasby, J. A., Artymiuk, P. J.,    Ceska, T. A. & Sayers, J. R. (2002). Interactions of mutant and    wild-type flap endonucleases with oligonucleotide substrates suggest    an alternative model of DNA binding. Proc Natl Acad Sci U S A 99,    8542-7.-   Deutscher, M. P. & Kornberg, A. (1969). Enzymatic synthesis of    deoxyribonucleic acid. XXIX. Hydrolysis of deoxyribonucleic acid    from the 5′ terminus by an exonuclease function of deoxyribonucleic    acid polymerase. J Biol Chem 244, 3029-37.-   Doherty, A. J., Serpell, L. C. & Ponting, C. P. (1996). The    helix-hairpin-helix DNA-binding motif: a structural basis for    non-sequence-specific recognition of DNA. Nucleic Acids Res 24,    2488-97.-   Garforth, S. J., Ceska, T. A., Suck, D. & Sayers, J. R. (1999).    Mutagenesis of conserved lysine residues in bacteriophage T5 5′-3′    exonuclease suggests separate mechanisms of endoand exonucleolytic    cleavage [In Process Citation]. Proc Natl Acad Sci U S A 96, 38-43.-   Goodsell, D. S., Kopka, M. L., Cascio, D. & Dickerson, R. E. (1993).    Crystal structure of CATGGCCATG and its implications for A-tract    bending models. Proc Natl Acad Sci U S A 90, 2930-4.-   Goulian, M., Richards, S. H., Heard, C. J. & Bigsby, B. M. (1990).    Discontinuous DNA synthesis by purified mammalian proteins    [published erratum appears in J Biol Chem 1990 Dec.    25;265(36):22569]. J Biol Chem 265, 18461-71.-   Gray, D. M., Hung, S. H. & Johnson, K. H. (1995). Absorption and    circular dichroism spectroscopy of nucleic acid duplexes and    triplexes. Methods Enzymol 246, 19-34.-   Habraken, Y., Sung, P., Prakash, L. & Prakash, S. (1993). Yeast    excision repair gene RAD2 encodes a single-stranded DNA    endonuclease. Nature 366, 365-8.-   Harrington, J. J. & Lieber, M. R. (1994a). The characterization of a    mammalian DNA structure-specific endonuclease. Embo J 13, 1235-46.-   Harrington, J. J. & Lieber, M. R. (1994b). Functional domains within    FEN-1 and RAD2 define a family of structure-specific endonucleases:    implications for nucleotide excision repair. Genes Dev 8, 1344-55.-   Hollingsworth, H. C. & Nossal, N. G. (1991). Bacteriophage T4    encodes an RNase H which removes RNA primers made by the T4 DNA    replication system in vitro. J Biol Chem 266, 1888-97.-   Hollis, T., Ichikawa, Y. & Ellenberger, T. (2000). DNA bending and a    flip-out mechanism for base excision by the helix-hairpin-helix DNA    glycosylase, Escherichia coli AlkA. Embo J 19, 758-66.-   Hosfield, D. J., Frank, G., Weng, Y., Tainer, J. A. & Shen, B.    (1998a). Newly discovered archaebacterial flap endonucleases show a    structure-specific mechanism for DNA substrate binding and catalysis    resembling human flap endonuclease-1. J Biol Chem 273, 27154-27161.-   Hosfield, D. J., Mol, C. D., Shen, B. & Tainer, J. A. (1998b).    Structure of the DNA repair and replication endonuclease and    exonuclease FEN-1: coupling DNA and PCNA binding to FEN-1 activity.    Cell 95, 135-146.-   Hou, Y. M., Zhang, X., Holland, J. A. & Davis, D. R. (2001). An    important 2′-OH group for an RNA-protein interaction. Nucleic Acids    Res 29, 976-85.-   Hwang, K. Y., Baek, K., Kim, H. Y. & Cho, Y. (1998). The crystal    structure of flap endonuclease-1 from Methanococcus jannaschii. Nat    Struct Biol 5, 707-713.-   Kaiser, M. W., Lyamicheva, N., Ma, W., Miller, C., Neri, B.,    Fors, L. & Lyamichev, V. I. (1999). A Comparison of Eubacterial and    Archaeal Structure-specific 5′- Exonucleases. J Biol Chem 274,    21387-21394.-   Kao, H. I., Henricksen, L. A., Liu, Y. & Bambara, R. A. (2002).    Cleavage Specificity of Saccharomyces cerevisiae Flap Endonuclease 1    Suggests a Double-Flap Structure as the Cellular Substrate. J Biol    Chem 277, 14379-89.-   Katahira, M., Miyanoiri, Y., Enokizono, Y., Matsuda, G., Nagata, T.,    Ishikawa, F. & Uesugi, S. (2001). Structure of the C-terminal    RNA-binding domain of hnRNP D0 (AUF1), its interactions with RNA and    DNA, and change in backbone dynamics upon complex formation with    DNA. J Mol Biol 311, 973-88.-   Kim, C. Y., Park, M. S. & Dyer, R. B. (2001). Human flap    endonuclease-1: conformational change upon binding to the flap DNA    substrate and location of the Mg2+ binding site. Biochemistry 40,    3208-14.-   Kim, Y., Eom, S. H., Wang, J., Lee, D. S., Suh, S. W. &    Steitz, T. A. (1995). Crystal structure of Thermus aquaticus DNA    polymerase. Nature 376, 612-616.-   Klenow, H. & Overgaard-Hansen, K. (1970). Proteolytic cleavage of    DNA polymerase from Escherichia coli B into an exonuclease unit and    a polymerase unit. FEBS Letters 6, 25-7.-   Lindahl, T., Gally, J. A. & Edelman, G. M. (1969). Deoxyribonuclease    IV: a new exonuclease from mammalian tissues. Proc Natl Acad Sci U S    A 62, 597-603.-   Lundquist, R. C. & Olivera, B. M. (1982). Transient generation of    displaced single-stranded DNA during nick translation. Cell 31,    53-60.-   Lyamichev, V., Brow, M. A. & Dahlberg, J. E. (1993).    Structure-specific endonucleolytic cleavage of nucleic acids by    eubacterial DNA polymerases. Science 260, 778-783.-   Lyamichev, V., Brow, M. A., Varvel, V. E. & Dahlberg, J. E. (1999).    Comparison of the 5′ Nuclease Activities of Taq DNA Polymerase and    Its Isolated Nuclease Domain. Proc Natl Acad Sci U S A Submitted.-   Matsui, E., Musti, K. V., Abe, J., Yamasaki, K., Matsui, I. &    Harata, K. (2002). Molecular structure and novel DNA binding sites    located in loops of flap endonuclease-1. J Biol Chem 277,    37840-37847.-   McGaughey, G. B., Gagne, M. & Rappe, A. K. (1998). pi-Stacking    interactions. Alive and well in proteins. J Biol Chem 273, 15458-63.-   Mueser, T. C., Nossal, N. G. & Hyde, C. C. (1996). Structure of    bacteriophage T4 RNase H, a 5′ to 3′ RNA-DNA and DNA-DNA exonuclease    with sequence similarity to the RAD2 family of eukaryotic proteins.    Cell 85, 1101-1112.-   Murante, R. S., Huang, L., Turchi, J. J. & Bambara, R. A. (1994).    The calf 5′- to 3′-exonuclease is also an endonuclease with both    activities dependent on primers annealed upstream of the point of    cleavage. J Biol Chem 269, 1191-1196.-   Murante, R. S., Rust, L. & Bambara, R. A. (1995). Calf 5′ to 3′    exo/endonuclease must slide from a 5′ end of the substrate to    perform structure-specific cleavage. J Biol Chem 270, 30377-30383.-   Murray, J. M., Tavassoli, M., al-Harithy, R., Sheldrick, K. S.,    Lehmann, A. R., Carr, A. M. & Watts, F. Z. (1994). Structural and    functional conservation of the human homolog of the    Schizosaccharomyces pombe rad2 gene, which is required for    chromosome segregation and recovery from DNA damage. Mol Cell Biol    14, 4878-4888.-   Noble, S. A., Fisher, E. F. & Caruthers, M. H. (1984).    Methylphosphonates as probes of protein-nucleic acid interactions.    Nucleic Acids Res 12, 3387-3404.-   O'Donovan, A., Davies, A. A., Moggs, J. G., West, S. C. &    Wood, R. D. (1994a). XPG endonuclease makes the 3′ incision in human    DNA nucleotide excision repair. Nature 371, 432-435.-   O'Donovan, A., Scherly, D., Clarkson, S. G. & Wood, R. D. (1994b).    Isolation of active recombinant XPG protein, a human DNA repair    endonuclease. J Biol Chem 269, 15965-8.-   Onufriev, A., Bashford, D. & Case, D. A. (200). Modification of the    Generalized Born Model Suitable for Macromolecules. J. Phys. Chem    104, 3712-3720.-   Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H. & Kraut, J.    (1996). Crystal structures of human DNA polymerase beta complexed    with DNA: implications for catalytic mechanism, processivity, and    fidelity. Biochemistry 35, 12742-61.-   Pritchard, C. E., Grasby, J. A., Hamy, F., Zacharek, A. M., Singh,    M., Kam, J. & Gait, M. J. (1994). Methylphosphonate mapping of    phosphate contacts critical for RNA recognition by the human    immunodeficiency virus tat and rev proteins. Nucleic Acids Res 22,    2592-600.-   Qiu, J., Bimston, D. N., Partikian, A. & Shen, B. (2002). Arginine    residues 47 and 70 of human flap endonuclease-1 are involved in DNA    substrate interactions and cleavage site determination. J Biol Chem    277, 24659-66.-   Reynaldo, L. P., Vologodskii, A. V., Neri, B. P. & Lyamichev, V. I.    (2000). The kinetics of oligonucleotide replacements. J Mol Biol    297, 511-520.-   Sawaya, M. R., Prasad, R., Wilson, S. H., Kraut, J. & Pelletier, H.    (1997). Crystal structures of human DNA polymerase beta complexed    with gapped and nicked DNA: evidence for an induced fit mechanism.    Biochemistry 36, 11205-15.-   Sayers, J. R. & Eckstein, F. (1990). Properties of overexpressed    phage T5 D15 exonuclease. Similarities with Escherichia coli DNA    polymerase I 5′-3′ exonuclease. J Biol Chem 265, 18311-7.-   Shao, X. & Grishin, N. V. (2000). Common fold in helix-hairpin-helix    proteins. Nucleic Acids Res 28, 2643-50.-   Shen, B., Nolan, J., Sklar, L. & Park, M. (1996). Essential amino    acids for substrate binding and catalysis of human flap    endonuclease 1. J. Biol. Chem. 271, 9173-9176.-   Shen, B., Nolan, J. P., Sklar, L. A. & Park, M. S. (1997).    Functional analysis of point mutations in human flap endonuclease-1    active site. Nucleic Acids Res 25, 3332-3338.-   Smith, S. A. & McLaughlin, L. W. (1997). Probing contacts to the DNA    backbone in the trp repressor-operator sequence-specific    protein-nucleic acid complex using diastereomeric methylphosphonate    analogues. Biochemistry 36, 6046-6058.-   Thayer, M. M., Ahern, H., Xing, D., Cunningham, R. P. &    Tainer, J. A. (1995). Novel DNA binding motifs in the DNA repair    enzyme endonuclease In crystal structure. Embo J. 14, 4108-4120.-   Tomky, L. A., Strauss-Soukup, J. K. & Maher, L. J., 3rd. (1998).    Effects of phosphate neutralization on the shape of the AP-1    transcription factor binding site in duplex DNA. Nucleic Acids Res    26, 2298-305.-   Tsui, V. & Case, D. A. (2000a). Molecular Dynamics Simulations of    Nucleic Acids with a Generalized Born Solvation Model. J. Am. Chem.    Soc. 122, 2489-2498.-   Tsui, V. & Case, D. A. (2000b). Theory and applications of the    generalized Born solvation model in macromolecular simulations.    Biopolymers 56, 275-291.-   Turchi, J. J. & Bambara, R. A. (1993). Completion of mammalian    lagging strand DNA replication using purified proteins. J Biol Chem    268, 15136-41.-   Weiner, S. J., Kollman, P. A., Case, D. A., Singh, U. C., Ghio, C.,    Alagona, G., Profeta, S. & Weiner, P. (1984). A New Force Field for    Molecular Mechanical Simulation of Nucleic Acids and Proteins. J.    Am. Chem. Soc 106, 765-784.-   Xu, Y., Derbyshire, V., Ng, K., Sun, X. C., Grindley, N. D. &    Joyce, C. M. (1997). Biochemical and mutational studies of the 5′-3′    exonuclease of DNA polymerase I of Escherichia coli. J Mol Biol 268,    284-302.-   Xu, Y., Potapova, O., Leschziner, A. E., Grindley, N. D. &    Joyce, C. M. (2001). Contacts between the 5′ nuclease of DNA    polymerase I and its DNA substrate. J Biol Chem 276, 30167-30177.

Example 70 Pfu FEN 1 R94A Substitution Mutant Cleaves MethylphosphonateSubstituted Probe

This example demonstrates that an engineered variant of Pfu FEN 1 inwhich the arginine at amino acid 94 was replaced with an alanineexhibits cleavage activity that differs from that of the wild typeenzyme. In particular, the R94A variant cleaves a substrate comprising amethylphosphonate-modified DNA probe.

Methylphosphonate substitutions are almost isosteric with phosphodiesterlinkages but unlike phosphodiester linkages are neutral and thereforecan be used to identify ionic interactions in protein/substratecomplexes without introducing steric clashes with the proteins(Dertinger, D., Dale, T. & Uhlenbeck, O. C. (2001). Modifying thespecificity of an RNA backbone contact. J Mol Biol 314, 649-654). Thebending angle estimated as 3.5 ° per methylphosphonate substitution(Tomky, L. A., Strauss-Soukup, J. K. & Maher, L. J., 3rd. (1998).Effects of phosphate neutralization on the shape of the AP-1transcription factor binding site in duplex DNA. Nucleic Acids Res 26,2298-305). is comparable to the intrinsic sequence-specific DNA bending(Goodsell, D. S., Kopka, M. L., Cascio, D. & Dickerson, R. E. (1993).Crystal structure of CATGGCCATG (SEQ ID NO:525) and its implications forA-tract bending models. Proc Natl Acad Sci U S A 90, 2930-4) and thermalflexibility of duplex DNA of ˜7 o per base pair estimated from itspersistence length (Cantor, C. R. & Schimmel, P. R. (1980). . InBiophysical Chemistry. Part III: The behavior of biologicalmacromolecules, pp. 1036-1037. W.H. Freeman and company, New York.).Substitution of a methyl group in place of a non-bridging oxygen in thephosphodiester linkage at a point of electrostatic contact with aprotein usually decreases the affinity of substrate binding (Dertinger &Uhlenbeck, ibid). This property of methylphosphonate modifications makesunnecessary, in most cases, the separation of Rp and Sp stereoisomers ofchemically introduced methylphosphonate linkages and justifies the useof their racemic mixtures.

Cleavage of such a racemic mixture of methylphosphonate-modified probesby the natural and modified enzymes was compared to determine whetherone or both isomers were suitable substrates for the enzymes. Invaderassay reaction mixtures were set up as follows:

Reaction component Stock concentration Final concentration Pfu FEN 1  10 nM  1 nM R94A variant of Pfu 44.7 μM 4.47 μM FEN1 Invader   2 μM500 nM oligo/probe/target mix 10 X DNA reaction 10X 1X buffer 1 Finalvolume 100 μl

Oligonucleotides were synthesized on an Expedite 8909 synthesizer(PerSeptive Biosystems) using standard phosphoramidite chemistry. Theupstream oligonucleotide, i.e. the INVADER oligonucleotide, was5′-CACTCCAGGGACGCGGACT-3′ (SEQ ID NO:526), the target oligonucleotidewas 5′-CTTTTACTCACCGCAGTTGGTCCGCGTCCCTGGAGTGTTC-3′ (SEQ ID NO:527), andthe downstream, probe oligonucleotide was 5′-TET-TTTTCAACTGCGGTGAG-3′(SEQ ID NO:528), where TET refers to tetracholorofluorescein, theunderlining indicates bases released following cleavage. The linkagebetween the bold bases was substituted with methylphosphonate in themodified substrate (Pd6). This modified substrate is denoted as1174-55-13 and forms a structure as diagrammed in FIG. 149. Reactionswere incubated at 50° C. for 4-5 hours, and 10 ìl aliquots weremonitored by denaturing PAGE (20%). The percentage of probeoligonucleotide cleaved was determined by quantitation of the cleavedand uncleaved probe gel bands as imaged on an FMBIO fluoroimager.

The results are presented in FIG. 158 and indicate that both enzymescleave the natural DNA substrate equally well. However, whereas the wildtype Pfu FEN 1 cleaved only 2% of the methylphosphonate-modified probe,the R94A substitution mutant cleaved approximately 50% of thesubstituted substrate.

The observation that the R94A enzyme variant cleaved approximately 50%of the P_(d)6 methylphosphonate substituted probe tends to indicate thatone stereoisomer and not the other is recognized as a suitable substrateby the mutant enzyme while neither isomer is cleaved efficiently by thenatural enzyme. While not limiting the methods of the present inventionto any particular mechanism, this result suggests that interaction ofthe methyl group at the P_(d)6 position of the probe with the alaninepresent at position 94 in the R94 mutant, likely a hydrophobicinteraction between the methyl and the alanine side group, can serve asalternative form of interaction, substituting for the phosphate-Arg94interaction between the natural substrate and enzyme.

It is contemplated that any number of the interactions that occur withina cleavage complex may be provided by alternative configurations of thecomponents of the complex. For example, it is contemplated thatcompositions, such as enzymes, that lack flap endonuclease activity maybe modified so as to comprise a functional group that confers thefunction of a Y33 residue, such that said compound is provided with flapendonuclease activity.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described methods and systems of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention which are obvious to those skilledin the relevant fields are intended to be within the scope of thefollowing claims.

1. A method for detecting the presence of a target nucleic acid moleculeby detecting non-target cleavage products, comprising: a) providing: aPfu FEN-1 nuclease comprising amino acid sequence SEQ ID NO:79 with theexception of amino acid position 94, wherein said Pfu FEN-1 comprises aY33 residue and a mutation in the R94 residue, wherein the mutation insaid R94 residue is R94A; a source of target nucleic acid, said targetnucleic acid comprising a first region and a second region, said secondregion downstream of and contiguous to said first region; a firstoligonucleotide, wherein a first portion of said first oligonucleotidecomprises at least one nucleotide analog and wherein said first portionis completely complementary to said first portion of said first targetnucleic acid, and; an aromatic moiety; b) combining said Pfu FEN-1nuclease, said target nucleic acid, said first oligonucleotide, and saidaromatic moiety under reaction conditions to form a cleavage complex,wherein said first portion of said first oligonucleotide is annealed tosaid first region of said target nucleic acid to form a duplex, whereinsaid Pfu FEN-1 nuclease recognizes said duplex, and wherein saidaromatic moiety interacts with said Y33 residue of said Pfu FEN-1nuclease, such that cleavage of said complex occurs to generate anon-target cleavage product; and c) detecting the cleavage of saidcleavage complex.
 2. The method of claim 1, wherein said interaction ofsaid aromatic moiety with said Y33 residue of said Pfu FEN-1 nucleasecomprises a stacking interaction.
 3. The method of claim 1, wherein saidaromatic moiety is a nucleotide.
 4. The method of claim 1, wherein saidaromatic moiety is a nucleotide analog.
 5. The method of claim 1,wherein a second oligonucleotide is provided, said secondoligonucleotide comprising a 3′ portion and a 5′ portion, wherein said5′ portion is completely complementary to said second region of saidtarget nucleic acid.
 6. The method of claim 5, wherein said 3′ portionof said second oligonucleotide comprises said aromatic moiety.
 7. Themethod of claim 1, wherein said nucleotide analog in said firstoligonucleotide comprises a methylphosphonate nucleotide.
 8. The methodof claim 1, wherein said nucleotide analog in said first oligonucleotidecomprises a 2′-O-methyl nucleotide.